US12337287B2 - Microfluidic devices - Google Patents

Microfluidic devices Download PDF

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Publication number
US12337287B2
US12337287B2 US15/480,739 US201715480739A US12337287B2 US 12337287 B2 US12337287 B2 US 12337287B2 US 201715480739 A US201715480739 A US 201715480739A US 12337287 B2 US12337287 B2 US 12337287B2
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Prior art keywords
droplets
droplet
fluid
channel
module
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US15/480,739
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US20170304785A1 (en
Inventor
Darren Roy Link
Michael Weiner
David Marran
Jonathan M. Rothberg
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Bio Rad Laboratories Inc
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Bio Rad Laboratories Inc
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Priority to US15/480,739 priority Critical patent/US12337287B2/en
Application filed by Bio Rad Laboratories Inc filed Critical Bio Rad Laboratories Inc
Assigned to RAINDANCE TECHNOLOGIES, INC. reassignment RAINDANCE TECHNOLOGIES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LINK, DARREN ROY, MARRAN, DAVID, ROTHBERG, JONATHAN M., WEINER, MICHAEL
Publication of US20170304785A1 publication Critical patent/US20170304785A1/en
Priority to US15/886,212 priority patent/US10639597B2/en
Priority to US15/996,236 priority patent/US20180280897A1/en
Priority to US15/996,246 priority patent/US10625220B2/en
Priority to US15/996,253 priority patent/US11351510B2/en
Assigned to BIO-RAD LABORATORIES, INC. reassignment BIO-RAD LABORATORIES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: RAINDANCE TECHNOLOGIES, INC.
Priority to US16/985,603 priority patent/US20200360876A1/en
Priority to US19/064,186 priority patent/US20250222413A1/en
Publication of US12337287B2 publication Critical patent/US12337287B2/en
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F23/00—Mixing according to the phases to be mixed, e.g. dispersing or emulsifying
    • B01F23/40—Mixing liquids with liquids; Emulsifying
    • B01F23/41—Emulsifying
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/30—Micromixers
    • B01F33/301—Micromixers using specific means for arranging the streams to be mixed, e.g. channel geometries or dispositions
    • B01F33/3011—Micromixers using specific means for arranging the streams to be mixed, e.g. channel geometries or dispositions using a sheathing stream of a fluid surrounding a central stream of a different fluid, e.g. for reducing the cross-section of the central stream or to produce droplets from the central stream
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0093—Microreactors, e.g. miniaturised or microfabricated reactors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • B03C5/005—Dielectrophoresis, i.e. dielectric particles migrating towards the region of highest field strength
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
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    • B03C5/02—Separators
    • B03C5/022—Non-uniform field separators
    • B03C5/026—Non-uniform field separators using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
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    • C12Q1/6813—Hybridisation assays
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y10T137/87587—Combining by aspiration
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y10T137/87571—Multiple inlet with single outlet
    • Y10T137/87652—With means to promote mixing or combining of plural fluids

Definitions

  • the present invention generally relates to systems and methods for the formation and/or control of fluidic species, and articles produced by such systems and methods. More particularly, the present invention relates to the development of high throughput microfluidic devices for precision fluid handling and use of such systems in various biological, chemical, or diagnostic assays.
  • Microfluidic systems have been described in a variety of contexts, typically in the context of miniaturized laboratory (e.g., clinical) analysis. Other uses have been described as well.
  • International Patent Application Publication No. WO 01/89788 describes multi-level microfluidic systems that can be used to provide patterns of materials, such as biological materials and cells, on surfaces.
  • Other publications describe microfluidic systems including valves, switches, and other components.
  • microfluidic devices Precision manipulation of streams of fluids with microfluidic devices is revolutionizing many fluid-based technologies. Networks of small channels are a flexible platform for the precision manipulation of small amounts of fluids.
  • the utility of such microfluidic devices depends critically on enabling technologies such as the microfluidic peristaltic pump, electrokinetic pumping, dielectrophoretic pump or electrowetting driven flow.
  • the assembly of such modules into complete systems provides a convenient and robust way to construct microfluidic devices.
  • virtually all microfluidic devices are based on flows of streams of fluids; this sets a limit on the smallest volume of reagent that can effectively be used because of the contaminating effects of diffusion and surface adsorption.
  • an electrically addressable emulsification system that combines compartmentalization and electrical manipulation, which allows for multi-step chemical processing, including analysis and sorting, to be initiated in confinement with vibrant timing and metering precision, for use in a variety of chemical, biological, and screening assays, in which the cost and time to perform such assays would be drastically reduced. It would also be desirable to develop a device using dielectrophoretic force (which does not rely on charge density) to manipulate droplets so that more than one electrical pondermotive function can be carried out following a significantly long delay from droplet formation.
  • the present invention provides substrates having individual fluid handling modules that can be combined into fluid processing systems so as to perform multi-step processing of isolated components, which is essential to perform biological, chemical and diagnostic applications, quickly, effectively and inexpensively.
  • the microfluidic substrates of the present invention can encapsulate reagents into droplets, which can be combined, analyzed, and sorted.
  • the present invention provides a microfluidic substrate.
  • the substrate can include a plurality of Microfluidic modules integrally arranged with each other so as to be in fluid communication.
  • the substrate can include, for example, (i) at least one inlet module having at least one inlet channel adapted to carry at least one dispersed phase fluid, (ii) at least one main channel adapted to carry at least one continuous phase fluid, wherein the inlet channel is in fluid communication with the main channel at a junction, wherein the junction includes a fluidic nozzle designed for flow focusing such that the dispersed phase fluid is immiscible with the continuous phase fluid and forms a plurality of highly uniform, monodisperse droplets in the continuous phase fluid.
  • the flow of the dispersed phase and continuous phase can be pressure driven, for example.
  • the dispersed phase e.g. droplets
  • the dispersed phase can be neutral or have no charge and these droplets can be manipulated (e.g., coalesced, sorted) within a electric field in the continuous phase fluid.
  • the inlet module can further include at least one self-aligning fluidic interconnect apparatus to connect the inlet channel to a means for introducing a sample fluid to the channel, wherein the apparatus forms a radial seal between the microfluidic substrate and the means for introducing sample.
  • the means can include, for example, a well or reservoir, which can be temperature controlled.
  • the well or reservoir can optionally include an acoustic actuator.
  • the microfluidic substrate can include one or more additional modules, including but not limited to, coalescence module, detection module, sorting module, collection module, waste module, delay module (e.g., heating and cooling modules), droplet spacing module, mixing module, UV-release module, division module and/or reordering module. These modules are in fluid communication with the main channel. There may be zero, one, or more of each of the modules.
  • the substrate can further include at least one coalescence module downstream from and in fluid communication with the inlet module via the main channel including a coalescence apparatus, wherein two or more droplets passing there through are coalesced to form a nanoreactor.
  • the substrate can further include at least one detection module downstream from and in fluid communication with the coalescence module.
  • the detection module can include, for example, a detection apparatus for evaluating the contents and/or characteristics of the nanoreactor.
  • the detection apparatus can include an optical or electrical detector.
  • the substrate can further include a sorting module downstream from and in fluid communication with the detection module.
  • the sorting module can include, for example, a sorting apparatus adapted to direct the nanoreactor into or away from a collection module in response to the contents or characterization of the nanoreactor evaluated in the detection module.
  • the channels in the sorting module can include an asymmetric bifurcation geometry or an asymmetric bifurcation flow.
  • the coalescence apparatus and the sorting apparatus can include one or more electrodes, or a patterned electrically conductive layer, which are capable of generating an electric field.
  • the electrodes can be made from electrically conductive materials, and can be integrally contained in one or more channels isolated from the main and inlet channels of the substrate.
  • the electrically conductive materials can be metal alloy components or organic materials.
  • the electrically conductive material can be an epoxy resin including one or more electrically conductive particles.
  • the electrically conductive particles can be silver particles.
  • the coalescence module can further include an expanded portion of the main channel between the electrodes to bring successive droplets into proximity, whereby the paired droplets are coalesced within the electric field.
  • the coalescence module can further include a narrowed portion of the main channel to center droplets within the main channel prior to the expanded portion of the main channel between the electrodes.
  • the channels of the microfluidic substrate can be coated with an anti-wetting or blocking agent for the dispersed phase.
  • the anti-wetting or blocking agent can include, for example, a silica primer layer followed by a perfluoroalkylalkylsilane compound, an amorphous soluble perfloropolymer, BSA, PEG-silane or fluorosilane.
  • the channels of the microfluidic substrate can include well-like indentations to slow, stop or react contents of droplets.
  • the substrate can further include a collection module connected to a means for storing a sample from the substrate and a waste module connected to a means for collecting a sample discarded from the substrate.
  • the means can be a well or reservoir, which can be temperature controlled.
  • the substrate can further include a delay module in fluid communication with the main channel downstream of the coalescence module and upstream of the detection module.
  • the delay module can be a delay line, serpentine channel, a buoyant hourglass, or an off-chip volume.
  • a serpentine channel is used to time delays less than 1 hour.
  • an off-chip volume is used to time delays longer than 1 hour.
  • the delay module can further include heating and cooling regions.
  • the substrate can further include a mixing module in fluid communication with the main channel downstream of the coalescence module and upstream of the detection module.
  • the substrate can further include a UV-release module in fluid communication with the main channel downstream of the inlet module and upstream of the coalescence module.
  • the substrate can further include a droplet spacing module in fluid communication with the main channel downstream of the inlet module to allow appropriate droplets to come with proximity for coalescence.
  • the continuous phase used in the channels of the microfluidic substrate can be a non-polar solvent such as, for example, a fluorocarbon oil.
  • the continuous phase can further include one or more additives such as a surfactant or fluorosurfactant in order to stabilize the droplets.
  • the fluorosurfactant can be a perfluorinated polyether, for example.
  • the dispersed phase of the microfluidic substrate can include a library of droplets of the same or different sizes (i.e., an emulsion stream) or a continuous aqueous stream.
  • the library of droplets can include, for example, a biological or chemical material such as tissues, cells, particles, proteins, antibodies, amino acids, nucleotides, small molecules, and pharmaceuticals.
  • the biological/chemical material can include a label such as a DNA tag, dye, a quantum dot or a radio frequency identification tag.
  • the library of library of droplets can include a label such as a change in viscosity, a change in opacity, a change in volume, a change in density, a change in pH, a change in temperature, a change in dielectric constant, a change in conductivity, a change in the amount of beads present in the droplets, a change in the amount of flocculent in the droplets, a change in the amount of a selected solvent within the droplets or the change in the amount of any measurable entity within the droplets, or combinations thereof.
  • a label can be detected by fluorescence polarization, fluorescence intensity, fluorescence lifetime, fluorescence energy transfer, pH, ionic content, temperature or combinations thereof.
  • the present invention also provides a microfluidic substrate including, for example, (i) at least one inlet module having at least one inlet channel adapted to carry at least one dispersed phase fluid; (ii) at least one main channel adapted to carry at least one continuous phase fluid, wherein the inlet channel is in fluid communication with the main channel at a junction, wherein the junction includes a fluidic nozzle designed for flow focusing such that the dispersed phase fluid is immiscible with the continuous phase fluid and forms a plurality of highly uniform, monodisperse droplets in the continuous phase fluid; (iii) at least one nanoreactor division module downstream from the inlet module wherein the main channel is divided into at least two division channels and the nanoreactor is split into at least two daughter nanoreactors; (iv) at least one second inlet channel adapted to carry at least one second dispersed phase fluid wherein the inlet channel is in fluid communication with at least one of the divisional channels at a junction, wherein the junction includes a fluidic nozzle designed for flow
  • the microfluidic substrate can also include a sorting module in proximity to and in fluid communication with the detection module, the sorting module including a sorting apparatus adapted to direct the droplet or nanoreactor into or away from a collection module in response to the contents or characterization of the droplet or nanoreactor evaluated in the detection module.
  • a sorting module in proximity to and in fluid communication with the detection module, the sorting module including a sorting apparatus adapted to direct the droplet or nanoreactor into or away from a collection module in response to the contents or characterization of the droplet or nanoreactor evaluated in the detection module.
  • the detection module can evaluate the contents of two nanoreactors or droplets in proximity and the sorting module can direct the droplets or nanoreactors into or away from a collection module in response to the ratio of the contents or characterization of the droplets or nanoreactors evaluated in the detection module.
  • the present invention also provides a method of producing microfluidic substrate including, for example, (i) providing a base plate, wherein the base plate comprises a flat surface; (ii) providing a master including the pattern of the channels and electrodes of a microfluidic substrate; (iii) providing a molding cavity, wherein the molding cavity comprises an opening for molding an elastomeric substrate; (iv) assembling the base plate, master and molding cavity, such that the master is placed between the base plate and molding cavity and wherein the master pattern is located directly under and aligned to the opening for molding an elastomeric substrate; (v) providing a top plate containing one or more sliding molding pins used to form one or more fluid and/or electrical interconnects; (vi) assembling the top plate onto the molding cavity of step d, such that the sliding molding pins contact points on the pattern of channels and electrodes on the master; (vi) introducing a liquid elastomeric polymer into the opening on the molding cavity such that it contacts the master; (vii) solidifying
  • the master is generated by photolithography, photolithography and converted to a durable metal master, micromachining or by rapid prototyping methods such as stereolithography.
  • the master can be a silicon or glass substrate patterned with photoresist.
  • the master is a silicon or glass substrate patterned with SU-8.
  • the elastomeric polymer can be a silicone elastomeric polymer.
  • the silicone elastreric polymer is polydimethylsiloxane.
  • the elastomeric polymer can be solidified by curing.
  • the elastomeric polymer can be treated with high intensity oxygen or air plasma to permit bonding to the compatible polymeric or non-polymeric media.
  • the polymeric and non-polymeric media can be glass, silicon, silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, or epoxy polymers.
  • FIG. 1 is an schematic illustrating the interacting modules of a microfluidic device of the present invention.
  • FIGS. 2 A-B show dual and single oil versions of the nozzle concept using a small ferrule for the nozzle section.
  • FIGS. 2 C-D show the same nozzles made directly out of small bore tubing (the “nozzle” runs the entire length of the tubing).
  • FIG. 3 shows the expansion of the nozzle ferrule concept shown in FIGS. 2 A and 2 B .
  • FIG. 4 shows the expansion of the nozzle section contained in the ferrule.
  • FIG. 5 A shows the operation of the nozzle in Aspiration Mode and 5 B shows the operation of the nozzle in Injection Mode.
  • FIG. 6 shows a reservoir based sample emulsification where the well is initially filled with a fluid with lower density than the sample to be introduced.
  • FIG. 7 illustrates a sample introduction when the sample is less dense than the fluid in the sample port, which is an alternative scheme used to introduce samples that are less dense than the oil used to emulsify the sample.
  • FIG. 8 illustrates a nozzle that formed directly into the fitting used to connect the collection syringe to a syringe tip (e.g. capillary tubing) in order to create a monodisperse emulsion directly from a library well.
  • Step 1 shows the aspiration of the sample can be accomplished by running the collection syringe in withdrawal mode at a flow rate (Q3) above the flow rate of the two oil syringes.
  • Step 2 shows the appropriate volume of sample loaded into the capillary tubing, and the capillary tubing would be removed from the sample well, an air bubble, and possibly a cleaning solution would be aspirated.
  • Step 3 shows when almost all of the sample has been emulsified, the collection syringe withdrawal rate would either be reduced below the oil flow rates, stopped, or set to infuse at some nominal rate.
  • FIGS. 9 A-C illustrate a two phase system where the reagent is injected on top of the 2nd, immiscible phase.
  • FIG. 9 A During injection, prior to transition from 1st phase to 2nd phase.
  • FIG. 9 B 2nd phase just entering the transfer lines.
  • FIG. 9 C 2nd phase has completely filled the transfer line and pushed the entire volume of reagent through the system.
  • FIG. 10 illustrates sandwiching an ultra-small volume of fluid (i.e., sub-nanoliter) between two solutions having different densities.
  • FIG. 11 illustrates possible interconnect designs for use with PDMS devices.
  • FIG. 12 illustrates self-alignment of fluidic interconnect
  • FIG. 13 illustrates the interconnects needed for each tube molded into a single monolithic self-aligned part.
  • FIG. 14 shows a schematic of a molding tool based on this concept.
  • the pins (orange) are captured within an elestomeric molded sleeve and a compression plate made from a rigid backer plate and foam rubber is used to apply gentle even pressure to the pins and generate the force needed to make the pins uniformly contact the master.
  • FIG. 15 is a schematic diagram of an improved coalescence module that shows an optional small constriction (neckdown) just before this expansion can be used to better align the droplets on their way into the coalescence point.
  • FIG. 16 illustrates that fluorescence polarization (FP) measures the tumbling rate of a compound in solution and is a function of it's volume (in most cases, volume is correlated with MW)
  • FIG. 17 shows the fluorescence polarization of three different compounds. Results of reading polarization in 18,000 drops containing 3 distinct species (FC, BTFC, and BTFC bound to SA). Ideal for reading results of drug screening assays, protein interactions, or DNA hybridization.
  • FIG. 18 A illustrates encoding a liquid solution using both overall fluorescence polarization and overall dye intensity within droplets
  • FIG. 18 B shows that multiple colors of fluorescence polarization and FI increases the number of possible labels. Ten intensity levels with ten fluorescence polarization levels on two colors yields 10,000 labels.
  • FIG. 19 illustrates FPcoding using dyes having different fluorescence lifetimes. These were made one element at a time, stored in a single syringe overnight and then loaded back on chip. The codes were made by using a ratio of two different dyes, one with a short lifetime and hence high FP and one with a long lifetime and correspondingly low FP. The mixtures have intermediate FP signals. The intensity is tuned by controlling the overall concentration of the two dyes.
  • FIG. 20 A- 20 D illustrate the sorting and/or splitting of droplets in accordance with another embodiment of the invention
  • FIG. 21 A-F shows the possible flow geometries used in an asymmetric sorting application.
  • FIGS. 22 A-E show the possible electrode geometries used in an asymmetric sorting application.
  • FIG. 22 A shows the design using sharp tipped electrodes.
  • FIG. 22 B shows broad tipped electrodes to increase the interaction time between the droplets and the electric field (the tips could be many drop diameters long).
  • FIG. 22 C shows electrodes straddling the collection line.
  • FIG. 22 D shows electrodes on opposite sides of the main channel.
  • FIG. 22 E shows an Asymmetric Electrode Pair (the asymmetry may be present on any of the other electrode pair layouts as well).
  • FIG. 23 shows a schematic of a device that split droplets, performs different experiments on the two daughter droplets and then reorders so that they pass sequential through the detector
  • FIG. 24 A shows geometric parameters defining the obstacle matrix.
  • FIG. 24 B shows three fluid streams.
  • FIG. 24 C shows a particle with a radius that is larger than lane 1 follows a streamline passing through the particle's center (black dot).
  • FIG. 25 shows high-resolution separation of fluorescent microspheres with diameters of 0.80 um (green), 0.90 um (red), and 1.03 um (yellow), with a matrix of varying gap size.
  • FIG. 26 is a schematic illustrating the separation by deterministic lateral displacement in an array of microposts, with an example row shift fraction of one-third.
  • FIG. 27 shows a dideoxynucleotide sequencing on a microfabricated chip. Shown is one embodiment for a DNA sequencing chip design. Template DNA and primers are combined at step ‘add 1’ and the reaction is incubated at 95° C. for a hot start (position 1). The reaction then cycles 20-30 times (position 2) before the addition of SAP and Exol at ‘add 2.’ The reaction is incubated at 37° C. for a pre-defined time-period and then the SAP and Exol enzymes are inactivated at 95° C. (position ‘4’). The SAP/Exol procedure degrades nucleotides and single-stranded DNA (primers) remaining after PCR. The universal sequencing primers, ddNTPs and buffers are added at ‘add 3,’ and the PCR sequencing reaction is allowed to proceed at position ‘5.’ The final reaction product is collected and can be stored off-chip.
  • FIG. 28 A shows a schematic of the TempliPhi amplification process using rolling circle amplification.
  • FIG. 28 B illustrates a transcription mediated reaction.
  • FIG. 28 C illustrates strand-displacement amplification.
  • FIG. 28 D shows a schematic diagram of helicase-dependent amplification.
  • FIG. 29 illustrates emulsion-based sample preparation, sample preparation and DNA sequencing. Random libraries of DNA fragments are generated by shearing an entire genome and isolating single DNA molecules by limiting dilution.
  • FIG. 30 shows one method for isolating antibodies on a microfluidic device.
  • FIG. 31 shows an alternate method for isolating antibodies on a microfluidic device.
  • FIG. 32 shows the method of the present invention for isolating antibodies on the microfluidic device.
  • the right panel is a diagram of individual steps proposed to amplify signal of interacting antibody and antigen.
  • the left panel is a schematic as would be designed for a chip to be used on microfluidic device.
  • FIG. 33 shows the genetic selection for full length antibody clones.
  • a genetic selection can be used to enrich for full-length antibody clones by transforming E. coli and selecting for clones able to grow on medium in which a suitable sugar is the only carbon source.
  • FIG. 37 shows the 8 non-renewable building blocks based on petroleum.
  • the microfluidic device of the present invention includes one or more analysis units.
  • An “analysis unit” is a microsubstrate, e.g., a microchip.
  • the terms microsubstrate, substrate, microchip, and chip are used interchangeably herein.
  • the analysis unit includes at least one inlet channel, at least one main channel, at least one inlet module, at least one coalescence module, and at least one detection module.
  • the analysis unit can further includes one or more sorting modules.
  • the sorting module can be in fluid communication with branch channels which are in fluid communication with one or more outlet modules (collection module or waste module). For sorting applications, at least one detection module cooperates with at least one sorting module to divert flow via a detector-originated signal.
  • modules and channels are in fluid communication with each other ‘and therefore may overlap; i.e., there may be no clear boundary where a module or channel begins or ends.
  • a plurality of analysis units of the invention may be combined in one device. The analysis unit and specific modules are described in further detail herein.
  • the dimensions of the substrate are those of typical microchips, ranging between about 0.5 cm to about 15 cm per side and about 1 micron to about 1 cm in thickness.
  • a substrate can be transparent and can be covered with a material having transparent properties, such as a glass coverslip, to permit detection of a reporter, for example, by an optical device such as an optical 25 microscope.
  • the material can be perforated for functional interconnects, such as fluidic, electrical, and/or optical interconnects, and sealed to the back interface of the device so that the junction of the interconnects to the device is leak-proof.
  • Such a device can allow for application of high pressure to fluid channels without leaking.
  • various components of the invention can be formed from solid materials, in which the channels can be formed via molding, micromachining, film deposition processes such as spin coating and chemical vapor deposition, laser fabrication, photolithographic techniques, etching methods including wet chemical or plasma processes, and the like. See, for example, Scientific American, 248:44-55, 1983 (Angell, et al). At least a portion of the fluidic system can be formed of silicone by molding a silicone chip. Technologies for precise and efficient formation of various fluidic systems and devices of the invention from silicone are known.
  • Various components of the systems and devices of the invention can also be formed of a polymer, for example, an elastomeric polymer such as polydimethylsiloxane (“PDMS”), polytetrafluoroethylene (“PTFE”) or Teflon®, or the like.
  • PDMS polydimethylsiloxane
  • PTFE polytetrafluoroethylene
  • Teflon® Teflon®
  • the channels of the invention can be formed, for example by etching a silicon chip using conventional photolithography techniques, or using a micromachining technology called “soft lithography” as described by Whitesides and Xia, Angewandte Chemie International Edition 37, 550 (1998). These and other methods may be used to provide inexpensive miniaturized devices, and in the case of soft lithography, can provide robust devices having beneficial properties such as improved flexibility, stability, and mechanical strength. When optical detection is employed, the invention also provides minimal light scatter from molecule, cell, small molecule or particle suspension and chamber material.
  • a base portion including a bottom wall and side walls can be formed from an opaque material such as silicone or PDMS, and a top portion can be formed from a transparent or at least partially transparent material, such as glass or a transparent polymer, for observation and/or control of the fluidic process.
  • Components can be coated so as to expose a desired chemical functionality to fluids that contact interior channel walls, where the base supporting material does not have a precise, desired functionality.
  • components can be formed as illustrated, with interior channel walls coated with another material.
  • Material used to form various components of the systems and devices of the invention may desirably be selected from among those materials that will not adversely affect or be affected by fluid flowing through the fluidic system, e.g., material(s) that is chemically inert in the presence of fluids to be used within the device.
  • Various components of the invention when formed from polymeric and/or flexible and/or elastomeric materials, and can be conveniently formed of a hardenable fluid, facilitating formation via molding (e.g. replica molding, injection molding, cast molding, etc.).
  • the hardenable fluid can be essentially any fluid that can be induced to solidify, or that spontaneously solidifies, into a solid capable of containing and/or transporting fluids contemplated for use in and with the fluidic network.
  • the hardenable fluid comprises a polymeric liquid or a liquid polymeric precursor (i.e. a “prepolymer”).
  • Suitable polymeric liquids can include, for example, thermoplastic polymers, thermoset polymers, or mixture of such polymers heated above their melting point.
  • a suitable polymeric liquid may include a solution of one or more polymers in a suitable solvent, which solution forms a solid polymeric material upon removal of the solvent, for example, by evaporation.
  • a suitable solvent such polymeric materials, which can be solidified from, for example, a melt state or by solvent evaporation, are well known to those of ordinary skill in the art.
  • a variety of polymeric materials, many of which are elastomeric, are suitable, and are also suitable for forming molds or mold masters, for embodiments where one or both of the mold masters is composed of an elastomeric material.
  • a non-limiting list of examples of such polymers includes polymers of the general classes of silicone polymers, epoxy polymers, and acrylate polymers.
  • Epoxy polymers are characterized by the presence of a three-membered cyclic ether group commonly referred to as an epoxy group, 1,2-epoxide, or oxirane.
  • diglycidyl ethers of bisphenol A can be used, in addition to compounds based on aromatic amine, triazine, and cycloaliphatic backbones.
  • Another example includes the well-known Novolac polymers.
  • Non-limiting examples of silicone elastomers suitable for use according to the invention include those formed from precursors including the chlorosilanes such as methylchlorosilanes, ethylchlorosilanes, phenylchlorosilanes, etc.
  • Silicone polymers are preferred, for example, the silicone elastomer polydimethylsiloxane.
  • Non-limiting examples of PDMS polymers include those sold under the trademark Sylgard by Dow Chemical Co., Midland, Mich., and particularly Sylgard 182, Sylgard 184, and Sylgard 186.
  • Silicone polymers including PDMS have several beneficial properties simplifying formation of the microfluidic structures of the invention. For instance, such materials are inexpensive, readily available, and can be solidified from a prepolymeric liquid via curing with heat.
  • PDMSs are typically curable by exposure of the prepolymeric liquid to temperatures of about, for example, about 65° C. to about 75° C. for exposure times of, for example, about an hour.
  • silicone polymers such as PDMS
  • PDMS polymethyl methacrylate copolymer
  • flexible (e.g., elastomeric) molds or masters can be advantageous in this regard.
  • the present invention provides improved methods of bonding PDMS to incompatible media.
  • Normal methods of bonding various materials (plastic, metals, etc) directly to materials such as PDMS, silicone, Teflon, and PEEK using traditional bonding practices (adhesives, epoxies, etc) do not Work well due to the poor adhesion of the bonding agent to materials such as PDMS.
  • Normal surface preparation by commercially available surface activators has not worked well in microfluidic device manufacturing. This problem is eliminated by treating the PDMS surface to be bonded with high intensity oxygen or air plasma. The process converts the top layer of PDMS to glass which bonds extremely well with normal adhesives.
  • Tests using this method to bond external fluid lines to PDMS using a UV-cure adhesive resulted in a bond that is stronger than the PDMS substrate, resulting in fracture of the PDMS prior to failure of the bond.
  • the present method combines high radiant flux, wavelength selection, and cure exposure time to significantly enhance the bond strength of the adhesive.
  • One advantage of forming structures such as microfluidic structures of the invention from silicone polymers, such as PDMS, is the ability of such polymers to be oxidized, for example by exposure to an oxygen-containing plasma such as an air plasma, so that the oxidized structures contain, at their surface, chemical groups capable of cross-linking to other oxidized silicone polymer surfaces or to the oxidized surfaces of a variety of other polymeric and non-polymeric materials.
  • an oxygen-containing plasma such as an air plasma
  • oxidized silicone such as oxidized PDMS can also be sealed irreversibly to a range of oxidized materials other than itself including, for example, glass, silicon, silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, and epoxy polymers, which have been oxidized in a similar fashion to the PDMS surface (for example, via exposure to an oxygen-containing plasma).
  • microfluidic structures of the invention or interior, fluid contacting surfaces
  • these surfaces can be much more hydrophilic than the surfaces of typical elastomeric polymers (where a hydrophilic interior surface is desired).
  • Such hydrophilic channel surfaces can thus be more easily filled and wetted with aqueous solutions than can structures comprised of typical, unoxidized elastomeric polymers or other hydrophobic materials.
  • a bottom wall is formed of a material different from one or more side walls or a top wall, or other components.
  • the interior surface of a bottom wall can comprise the surface of a silicon wafer or microchip, or other substrate.
  • Other components can, as described above, be sealed to such alternative substrates.
  • a component comprising a silicone polymer e.g. PDMS
  • the substrate may be selected from the group of materials to which oxidized silicone polymer is able to irreversibly seal (e.g., glass, silicon, silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, epoxy polymers, and glassy carbon surfaces which have been oxidized).
  • other sealing techniques can be used, as would be apparent to those of ordinary skill in the art, including, but not limited to, the use of separate adhesives, thermal bonding, solvent bonding, ultrasonic welding, etc.
  • the microfluidic substrates of the present invention include channels that form the boundary for a fluid.
  • a “channel,” as used herein, means a feature on or in a substrate that at least partially directs the flow of a fluid.
  • the channel may be formed, at least in part, by a single component, e.g., an etched substrate or molded unit.
  • the channel can have any cross-sectional shape, for example, circular, oval, triangular, irregular, square or rectangular (having any aspect ratio), or the like, and can be covered or uncovered (i.e., open to the external environment surrounding the channel).
  • at least one portion of the channel can have a cross-section that is completely enclosed, and/or the entire channel may be completely enclosed along its entire length with the exception of its inlet and outlet.
  • An open channel generally will include characteristics that facilitate control over fluid transport, e.g., structural characteristics (an elongated indentation) and/or physical or chemical characteristics (hydrophobicity vs. hydrophilicity) and/or other characteristics that can exert a force (e.g., a containing force) on a fluid.
  • the fluid within the channel may partially or completely fill the channel.
  • the fluid may be held or confined within the channel or a portion of the channel in some fashion, for example, using surface tension (e.g., such that the fluid is held within the channel within a meniscus, such as a concave or convex meniscus).
  • some (or all) of the channels may be of a particular size or less, for example, having a largest dimension perpendicular to fluid flow of less than about 5 mm, less than about 2 mm, less than about 1 mm, less than about 500 microns, less than about 200 microns, less than about 100 microns, less than about 60, less than about 50 microns, less than about 40 microns, less than about 30 microns, less than about 25 microns, less than about 10 microns, less than about 3 microns, less than about 1 micron, less than about 300 nm, less than about 100 nm, less than about 30 nm, or less than about 10 nm or less in some cases.
  • larger channels, tubes, etc. can be used to store fluids in bulk and/or deliver a fluid to the channel.
  • the channel is a capillary.
  • the dimensions of the channel may be chosen such that fluid is able to freely flow through the channel, for example, if the fluid contains cells.
  • the dimensions of the channel may also be chosen, for example, to allow a certain volumetric or linear flow rate of fluid in the channel.
  • the number of channels and the shape of the channels can be varied by any method known to those of ordinary skill in the art. In some cases, more than one channel or capillary may be used. For example, two or more channels may be used, where they are positioned inside each other, positioned adjacent to each other, etc.
  • the channels of the device are preferably square, with a diameter between about 2 microns and 1 mm. This geometry facilitates an orderly flow of droplets in the channels.
  • the volume of the detection module in an analysis device is typically in the range of between about 0.1 picoliters and 500 nanoliters.
  • a “main channel” is a channel of the device of the invention which permits the flow of molecules, cells, small molecules or particles past a coalescence module for coalescing one or more droplets, a detection module for detection (identification) or measurement of a droplet and a sorting module, if present, for sorting a droplet based on the detection in the detection module.
  • the main channel is typically in fluid communication with the coalescence, detection and/or sorting modules, as well as, an inlet channel of the inlet module.
  • the main channel is also typically in fluid communication with an outlet module and optionally with branch channels, each of which may have a collection module or waste module. These channels permit the flow of molecules, cells, small molecules or particles out of the main channel.
  • the microfluidic substrate can also comprise one or more fluid channels to inject or remove fluid in between droplets in a droplet stream for the purpose of changing the spacing between droplets.
  • fluid stream or “fluidic stream” refers to the flow of a fluid, typically generally in a specific direction.
  • the fluidic stream may be continuous and/or discontinuous.
  • a “continuous” fluidic stream is a fluidic stream that is produced as a single entity, e.g., if a continuous fluidic stream is produced from a channel, the fluidic stream, after production, appears to be contiguous with the channel outlet.
  • the continuous fluidic stream is also referred to as a continuous phase fluid or carrier fluid.
  • the continuous fluidic stream may be laminar, or turbulent in some cases.
  • a “discontinuous” fluidic stream is a fluidic stream that is not produced as a single entity.
  • the discontinuous fluidic stream is also referred to as the dispersed phase fluid or sample fluid.
  • a discontinuous fluidic stream may have the appearance of individual droplets, optionally surrounded by a second fluid.
  • a “droplet,” as used herein, is an isolated portion of a first fluid that completely surrounded by a second fluid.
  • emulsion refers to a preparation of one liquid distributed in small globules (also referred to herein as drops, droplets or NanoReactors) in the body of a second liquid.
  • the first and second fluids are immiscible with each other.
  • the discontinuous phase can be an aqueous solution and the continuous phase can a hydrophobic fluid such as an oil. This is termed a water in oil emulsion.
  • the emulsion may be a oil in water emulsion.
  • the first liquid, which is dispersed in globules is referred to as the discontinuous phase
  • the second liquid is referred to as the continuous phase or the dispersion medium.
  • the continuous phase can be an aqueous solution and the discontinuous phase is a hydrophobic fluid, such as an oil (e.g., decane, tetradecane, or hexadecane).
  • a hydrophobic fluid such as an oil (e.g., decane, tetradecane, or hexadecane).
  • the droplets or globules of oil in an oil in water emulsion are also referred to herein as “micelles”, whereas globules of water in a water in oil emulsion may be referred to as “reverse micelles”.
  • the fluidic droplets may each be substantially the same shape and/or size.
  • the shape and/or size can be determined, for example, by measuring the average diameter or other characteristic dimension of the droplets.
  • the “average diameter” of a plurality or series of droplets is the arithmetic average of the average diameters of each of the droplets. Those of ordinary skill in the art will be able to determine the average diameter (or other characteristic dimension) of a plurality or series of droplets, for example, using laser light scattering, microscopic examination, or other known techniques.
  • the diameter of a droplet, in a non-spherical droplet is the mathematically-defined average diameter of the droplet, integrated across the entire surface.
  • the average diameter of a droplet may be, for example, less than about 1 mm, less than about 500 micrometers, less than about 200 micrometers, less than about 100 micrometers, less than about 75 micrometers, less than about 50 micrometers, less than about 25 micrometers, less than about 10 micrometers, or less than about 5 micrometers in some cases.
  • the average diameter may also be at least about 1 micrometer, at least about 2 micrometers, at least about 3 micrometers, at least about 5 micrometers, at least about 10 micrometers, at least about 15 micrometers, or at least about 20 micrometers in certain cases.
  • NanoReactor and its plural encompass the terms “droplet”, “nanodrop”, “nanodroplet”, “microdrop” or “microdroplet” as defined herein, as well as an integrated system for the manipulation and probing of droplets, as described in detail herein.
  • Nanoreactors as described herein can be 0.1-1000 ⁇ m (e.g., 0.1, 0.2 . . . 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 . . . 1000), or any size within in this range. Droplets at these dimensions tend to conform to the size and shape of the channels, while maintaining their respective volumes. Thus, as droplets move from a wider channel to a narrower channel they become longer and thinner, and vice versa.
  • the microfluidic substrate of this invention most preferably generate round, monodisperse droplets.
  • the droplets can have a diameter that is smaller than the diameter of the microchannel; i.e., preferably 15 to 100 ⁇ m when cells are used; or 10 to 75 ⁇ m when reagents or other chemical or biological agents are used; or 100 to 1000 ⁇ m when droplets are used for sequencing reactions such that droplets will be removed and dispensed into other collection apparatuses, such as microtiter plates or utilized in sequencing devices.
  • Monodisperse droplets are particularly preferably, e.g., in high throughput devices and other embodiments where it is desirable to generate droplets at high frequency and of high uniformity.
  • the droplet forming liquid is typically an aqueous buffer solution, such as ultrapure water (e.g., 18 mega-ohm resistivity, obtained, for example by column chromatography), 10 mM Tris HCl and 1 mM EDTA (TE) buffer, phosphate buffer saline (PBS) or acetate buffer. Any liquid or buffer that is physiologically compatible with the population of molecules, cells or particles to be analyzed and/or sorted can be used.
  • the fluid passing through the main channel and in which the droplets are formed is one that is immiscible with the droplet forming fluid.
  • the fluid passing through the main channel can be a non-polar solvent, decane (e g., tetradecane or hexadecane), fluorocarbon oil, silicone oil or another oil (for example, mineral oil).
  • the dispersed phase fluid may also contain biological/chemical material (e.g., molecules, cells or other particles) for combination, analysis and/or sorting in the device.
  • the droplets of the dispersed phase fluid can contain more than one particle or can contain no more than one particle.
  • each droplet preferably contains, on average, no more than one cell.
  • each droplet may contain, on average, at least 1000 cells.
  • the droplets can be detected and/or sorted according to their contents.
  • the concentration (i.e., number) of molecules, cells or particles in a droplet can influence sorting efficiently and therefore is preferably optimized.
  • the sample concentration should be dilute enough that most of the droplets contain no more than a single molecule, cell or particle, with only a small statistical chance that a droplet will contain two or more molecules, cells or particles. This is to ensure that for the large majority of measurements, the level of reporter measured in each droplet as it passes through the detection module corresponds to a single molecule, cell or particle and not to two or more molecules, cells or particles.
  • the parameters which govern this relationship are the volume of the droplets and the concentration of molecules, cells or particles in the sample solution.
  • [cell]” is the concentration of molecules, cells or particles in units of number of molecules, cells or particles per cubic micron ( ⁇ m 3 ), and V is the volume of the droplet in units of ⁇ m 3 .
  • the maximum tolerable P ⁇ 2 depends on the desired “purity” of the sorted sample.
  • the “purity” in this case refers to the fraction of sorted molecules, cells or particles that posses a desired characteristic (e.g., display a particular antigen, are in a specified size range or are a particular type of molecule, cell or particle).
  • the purity of the sorted sample is inversely proportional to P ⁇ 2 .
  • P ⁇ 2 a relatively high
  • FIG. 3 shows the expansion of the nozzle ferrule concept shown in FIGS. 2 A and 2 B .
  • the tube based nozzles ( FIG. 2 C, 2 D ) function identically to this, except the “nozzle” runs the entire length of the tube instead of having a short transition.
  • the ability to form droplets is identical in both cases.
  • FIG. 4 shows the expansion of the nozzle section contained in the ferrule. The tee design in FIG.
  • the sample tip is connected to a pump capable of driving the sample into the device, it could be started up as the tip is inserted into the device ( 7 b - c ).
  • the device could be run identically to the “normal” operation of our devices, including having the “transport to waste” line ( 7 e ) not connected to a pump.
  • the sample tip loading pump is not capable of accurately forcing the flow (i.e. not connected to a suitable pump)
  • the back end of the tip could be connected to a valve that would open to either atmospheric pressure (or possibly a pressurized gas supply) when the tip is fully inserted into the port.
  • FIG. 7 also shows another possible configuration of the aspiration probe assembly used for the device in FIG. 6 .
  • the nozzle can be formed through using small bore tubing (glass, Teflon®, PEEK tubing or capillaries) or micro-fabrication or molding processes such as PDMS soft lithography, glass etching, hot embossing, or similar high resolution fabrication technology.
  • small bore tubing glass, Teflon®, PEEK tubing or capillaries
  • micro-fabrication or molding processes such as PDMS soft lithography, glass etching, hot embossing, or similar high resolution fabrication technology.
  • the present apparatus can be readily adapted for clinical applications or work where cross contamination must be eliminated, since the region from the nozzle to the syringe are isolated from the sample stream (e.g., the oil wets these surfaces and keeps the sample from directly contacting aqueous sample).
  • the aspiration tip can be designed as a disposable item (like a robotic sampler aspiration tips) and automatically replaced between samples.
  • Multiple nozzle/syringe pairs can be operated in parallel, thus increasing throughput. This allows simultaneous sampling of multiple wells/samples during a single process step. Each sample can be collected into a separate syringe.
  • sample droplet emulsions and emulsion libraries “off chip” are described in Example 1.
  • the present invention provides compositions and methods which eliminates the problems associated with dead volume and reagent waste when working with extremely small volumes of reagents.
  • the primary reagents is combined with a second, immiscible phase in the storage container (e.g. a syringe or other reservoir).
  • a second, immiscible phase in the storage container (e.g. a syringe or other reservoir).
  • This second phase is used to push the entire amount of the first phase into the system with no significant losses. More specifically, when two immiscible fluids are combined in a reservoir, the two fluids will tend to separate into layers as long as the densities of the materials are different. If the fluid of interest (e.g., sample fluid) is closest to the exit of the reservoir, it will be the first to leave when the reservoir is emptied (the exit can be on either the top or bottom, depending on the density difference). Once the reagent has been pumped out of the reservoir, the second phase will follow.
  • the fluid of interest e.g., sample fluid
  • This second phase will then push the first phase completely through the system without any sample fluid loss.
  • oil and water the reagent
  • the syringe would be combined in a syringe. If the oil is denser than the water, the syringe would be oriented with its exit face up, if the oil were less dense, then the syringe would be face down. The oil would be chosen such that the materials of interest in the reagent are not soluble in the oil phase.
  • FIG. 9 is one example of this approach when a syringe is used as the reservoir and the second phase is denser than the reagent phase. If the reagent were more dense, then the syringe orientation would be reversed (i.e. the exit would be facing downward in the figure).
  • FIG. 9 shows a two phase system where the reagent is injected on top of the second, immiscible phase: (A) During injection, prior to transition from first phase to second phase, (B) second phase just entering the transfer line, (C) second phase has completely filled the transfer line and pushed the entire volume of reagent through the system.
  • a sample solution is sandwiched between two immiscible liquids, wherein one liquid has a density greater than the sample density, and the second liquid has a density less than the sample density.
  • the sample density 1.0
  • the sample can be layered between perflourocarbon oil (density 1.8) and mineral oil (density 0.914).
  • perflourocarbon oil density 1.8
  • mineral oil density 0.914
  • the sample then rises to the injection point after the mineral oil. It is further contemplated that the methods disclosed herein would also work for gases.
  • the gases and/or liquids can be miscible, but of different densities such that they are layered on top each other in a manner that prevents their mixing.
  • proteins can be either trapped within, or attached to the gel or polymer matrix. If attached, it can be through a covalent linkage or through an affinity tag, for example his6 or avi-tag.
  • the proteins can be added to droplets containing gel or polymer reagent, or they can be formulated along with the gel or polymerization reagent. Variations that include both are also possible.
  • the protein can be added to the droplets. Additionally, it is possible to add DNA to the droplet and allow in vitro transcription/translation to synthesize the protein.
  • the droplets may be coated with a surfactant.
  • Preferred surfactants that may be added to the continuous phase fluid include, but are not limited to, surfactants such as sorbitan-based carboxylic acid esters (e.g., the “Span” surfactants, Fluka Chemika), including sorbitan monolaurate (Span 20), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60) and sorbitan monooleate (Span 80), and perfluorinated polyethers (e.g., DuPont Krytox 157 FSL, FSM, and/or FSH).
  • surfactants such as sorbitan-based carboxylic acid esters (e.g., the “Span” surfactants, Fluka Chemika), including sorbitan monolaurate (Span 20), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60) and sorbitan monooleate (Span 80), and perfluorin
  • ionic surfactants such as sodium dodecyl sulfate (SDS) may also be used.
  • SDS sodium dodecyl sulfate
  • surfactants are generally less preferably for many embodiments of the invention.
  • a water soluble surfactant such as SDS may denature or inactivate the contents of the droplet.
  • the carrier fluid can be an oil (e.g., decane, tetradecane or hexadecane) or fluorocarbon oil that contains a surfactant (e.g., a non-ionic surfactant such as a Span surfactant) as an additive (preferably between about 0.2 and 5% by volume, more preferably about 2%).
  • a surfactant e.g., a non-ionic surfactant such as a Span surfactant
  • a user can preferably cause the carrier fluid to flow through channels of the microfluidic device so that the surfactant in the carrier fluid coats the channel walls.
  • the fluorosurfactant can be prepared by reacting the perflourinated polyether DuPont Krytox 157 FSL, FSM, or FSH with aqueous ammonium hydroxide in a volatile fluorinated solvent.
  • the solvent and residual water and ammonia can be removed with a rotary evaporator.
  • the surfactant can then be dissolved (e.g., 2.5 wt %) in a fluorinated oil (e.g., Flourinert (3M)), which then serves as the continuous phase of the emulsion.
  • a fluorinated oil e.g., Flourinert (3M)
  • the invention can use pressure drive flow control, e.g., utilizing valves and pumps, to manipulate the flow of cells, particles, molecules, enzymes or reagents in one or more directions and/or into one or more channels of a microfluidic device.
  • pressure drive flow control e.g., utilizing valves and pumps
  • other methods may also be used, alone or in combination with pumps and valves, such as electro-osmotic flow control, electrophoresis and dielectrophoresis (Fulwyer, Science 156, 910 (1974); Li and Harrison, Analytical Chemistry 69, 1564 (1997); Fiedler, et al. Analytical Chemistry 70, 1909-1915 (1998); U.S. Pat. No. 5,656,155).
  • Positive displacement pressure driven flow is a preferred way of controlling fluid flow and dielectrophoresis is a preferred way of manipulating droplets within that flow.
  • the pressure at the inlet module can also be regulated by adjusting the pressure on the main and sample inlet channels, for example, with pressurized syringes feeding into those inlet channels.
  • the size and periodicity of the droplets generated may be regulated.
  • a valve may be placed at or coincident to either the inlet module or the sample inlet channel connected thereto to control the flow of solution into the inlet module, thereby controlling the size and periodicity of the droplets.
  • Periodicity and droplet volume may also depend on channel diameter, the viscosity of the fluids, and shear pressure.
  • electro-osmosis is believed to produce motion in a stream containing ions e.g. a liquid such as a buffer, by application of a voltage differential or charge gradient between two or more electrodes. Neutral (uncharged) molecules or cells can be carried by the stream. Electro-osmosis is particularly suitable for rapidly changing the course, direction or speed of flow. Electrophoresis is believed to produce movement of charged objects in a fluid toward one or more electrodes of opposite charge, and away from one on or more electrodes of like charge. Where an aqueous phase is combined with an oil phase, aqueous droplets are encapsulated or separated from each other by oil.
  • the oil phase is not an electrical conductor and may insulate the droplets from the electro-osmotic field.
  • electro-osmosis may be used to drive the flow of droplets if the oil is modified to carry or react to an electrical field, or if the oil is substituted for another phase that is immiscible in water but which does not insulate the water phase from electrical fields.
  • Dielectrophoresis is believed to produce movement of dielectric objects, which have no net charge, but have regions that are positively or negatively charged in relation to each other.
  • dielectric polarizability of the particles and the suspending medium dielectric particles will move either toward the regions of high field strength or low field strength.
  • the polarizability of living cells depends on their composition, morphology, and phenotype and is highly dependent on the frequency of the applied electrical field.
  • cells of different types and in different physiological states generally possess distinctly different dielectric properties, which may provide a basis for cell separation, e.g., by differential dielecrophoretic forces.
  • the polarizability of droplets also depends upon their size, shape and composition. For example, droplets that contain salts can be polarized. According to formulas provided in Fiedler, et al. Analytical Chemistry 70, 1909-1915 (1998), individual manipulation of sine, droplets requires field differences (inhomogeneities) with dimensions close to the droplets.
  • dielectrophoretic force gradient means a dielectrophoretic force is exerted on an object in an electric field provided that the object has a different dielectric constant than the surrounding media. This force can either pull the object into the region of larger field or push it out of the region of larger field. The force is attractive or repulsive depending respectively on whether the object or the surrounding media has the larger dielectric constant.
  • Manipulation is also dependent on permittivity (a dielectric property) of the droplets and/or particles with the suspending medium.
  • permittivity a dielectric property
  • polymer particles, living cells show negative—dielectrophoresis at high-field frequencies in water.
  • dielectrophoretic forces experienced by a latex sphere in a 0.5 MV/m field (10 V for a 20 micron electrode gap) in water are predicted to be about 0.2 piconewtons (pN) for a 3.4 micron latex sphere to 15 pN for a 15 micron latex sphere (Fiedler, et al. Analytical Chemistry 70, 1909-1915 (1998)).
  • Radiation pressure can also be used in the invention to deflect and move objects, e.g. droplets and particles (molecules, cells, particles, etc.) contained therein, with focused beams of light such as lasers.
  • Flow can also be obtained and controlled by providing a pressure differential or gradient between one or more channels of a device or in a method of the invention.
  • Molecules, cells or particles can be moved by direct mechanical switching, e.g., with on-off valves or by squeezing the channels. Pressure control may also be used, for example, by raising or lowering an output well to change the pressure inside the channels on the chip. See, e.g., the devices and methods described U.S. Pat. No. 6,540,895. These methods and devices can further be used in combination with the methods and devices described in pending U.S. Patent Application Publication No. 20010029983 and 20050226742. Different switching and flow control mechanisms can be combined on one chip or in one device and can work independently or together as desired.
  • the microfluidic device of the present invention includes one or more inlet modules.
  • An “inlet module” is an area of a microfluidic substrate device that receives molecules, cells, small molecules or particles for additional coalescence, detection and/or sorting.
  • the inlet module can contain one or more inlet channels, wells or reservoirs, openings, and other features which facilitate the entry of molecules, cells, small molecules or particles into the substrate.
  • a substrate may contain more than one inlet module if desired. Different sample inlet channels can communicate with the main channel at different inlet modules. Alternately, different sample inlet channels can communication with the main channel at the same inlet module.
  • the inlet module is in fluid communication with the main channel.
  • the inlet module generally comprises a junction between the sample inlet channel and the main channel such that a solution of a sample (i.e., a fluid containing a sample such as molecules, cells, small molecules (organic or inorganic) or particles) is introduced to the main channel and forms a plurality of droplets.
  • a sample i.e., a fluid containing a sample such as molecules, cells, small molecules (organic or inorganic) or particles
  • the sample solution can be pressurized.
  • the sample inlet channel can intersect the main channel such that the sample solution is introduced into the main channel at an angle perpendicular to a stream of fluid passing through the main channel.
  • the sample inlet channel and main channel intercept at a T-shaped junction; i.e., such that the sample inlet channel is perpendicular (90 degrees) to the main channel.
  • the sample inlet channel can intercept the main channel at any angle, and need not introduce the sample fluid to the channel at an angle that is perpendicular to that flow.
  • the angle between intersecting channels is in the range of from about 60 to about 120 degrees. Particular exemplary angles are 45, 60, 90, and 120 degrees.
  • Embodiments of the invention are also provided in which there are two or more inlet modules introducing droplets of samples into the main channel.
  • a first inlet module may introduce droplets of a first sample into a flow of fluid in the main channel and a second inlet module may introduce droplets of a second sample into the flow of fluid in main channel, and so forth.
  • the second inlet module is preferably downstream from the first inlet module (e.g., about 30 ⁇ m).
  • the fluids introduced into the two or more different inlet modules can comprise the same fluid or the same type of fluid (e.g., different aqueous solutions).
  • the tubing side of the interconnect can be mounted into a retaining block that provides precise registration of the tubing, while the microfluidic device can be positioned accurately in a carrier that the retaining block would align and clamp to.
  • the total dead volume associated with these designs would be critically dependent on how accurately the two mating surfaces could be positioned relative to each other.
  • the maximum force required to maintain the seal would be limited by the exact shape and composition of the sealing materials as well as the rigidity and strength of the device itself.
  • the shapes of the mating surfaces can be tailored to the minimal leakage potential, sealing force required, and potential for misalignment.
  • the single ring indicated in can be replaced with a series of rings of appropriate cross-sectional shape.
  • the present invention includes one or more inlet modules comprising self-aligning fluidic interconnects proximate to one or more inlet channels to improve the efficiency of sample loading and/or injection.
  • the present invention proposes the use of small interconnects based on creating a radial seal instead of a face seal between the microfluidic device and interconnect.
  • the inserted interconnect would have a larger diameter than the mating feature on the device.
  • the stretching of the chip would provide the sealing force needed to make a leak-free seal between the external fluid lines and the microfluidic device.
  • FIG. 11 details design possibilities for making this seal.
  • the external interconnect In order to handle instrument and chip manufacturing tolerances, the external interconnect must be self aligning and the “capture radius” of the molded hole must be large enough to reliably steer the interconnect to the sealing surfaces.
  • FIG. 12 shows that the entrance to the molded hole is large enough to guarantee capture but tapers down to the sealing surfaces.
  • the external interconnect could be made directly out of the tubing leading up to the microfluidic substrate, thus eliminating potential leak points and unswept volumes. As seen in FIG. 12 , the interconnect is surrounded by the substrate interconnects or “chip dock” for most of its length to make certain it is held within the tolerance stack-up of the system.
  • the external interconnect is made from a hard but flexible material such as 1/32′′ PEEK tubing.
  • the features in the microfluidic device can be molded directly into it during the manufacturing process, while the inserted seals can be molded/machined directly onto the tubing ends or molded as individual pieces and mechanically fastened to the tubing.
  • the retaining ferrule shown in FIG. 12 would be attached during manufacturing and provide good absolute referencing of the tube length.
  • the ferrule could be an off-the-shelf component or a custom manufactured part and be made from, for example, a polymer, an elastomer, or a metal.
  • the tubing end could be tapered on the end (top most diagram) or squared off (the figure above). The specific shape of the end will be controlled by how easily the microfluidic device will gall during insertion.
  • the present invention also provides methods of direct molding of fluidic interconnects into a microfluidic device.
  • Development of a commercial microfluidic platform requires a simple, reliable fluidic interconnect in order to reduce the chance of operator error and leaks. Molding these interconnects directly into the microfluidic device requires precise alignment of the molding pins to 30 the patterned shim (the “master” manufactured from Silicon/photoresist or made from some metal) used to form the microfluidic and electrical channels.
  • the master manufactured from Silicon/photoresist or made from some metal
  • the extreme tolerances required when molding with low viscosity elastomer such as PDMS requires near perfect sealing of the pin face to the mast r, while still accommodating imperfections in the master and assembly of the molding tool.
  • the present invention provides a precise and repeatable method of molding of interconnects while accommodating the imperfections in the molding process by introducing movable pins captured in an elastomeric sleeve molded directly into the tool.
  • the tool In order to effectively produce at relatively low volume and be able to inexpensively prototype devices, the tool must be able to use masters generated using standard photolithographic processes (e.g. silicon wafers patterned with SU 8).
  • FIG. 14 shows a schematic of a molding tool based on this concept.
  • the pins (orange) are captured within an elestomeric molded sleeve.
  • a compression plate made from a rigid backer plate and foam rubber is used to apply gentle even pressure to the pins and generate the force needed to make the pins uniformly contact the master.
  • the molded sleeve was found to be necessary to consistently prevent the uncured elastomer from penetrating the region between the pin and the top plate.
  • Early designs used pins captured in tight clearance holes, and the pins would frequently bind in place (even with lubricant), preventing smooth motion of the pins and improper contact with the master.
  • the well or reservoir of the inlet module further include an acoustic actuator.
  • an acoustic actuator To obtain one droplet comprising a single element of a specific biological/chemical material (e.g., a cell), separation of biological/chemical material, and uniformity of the number density of biological/chemical materials in a microfluidic channel is desirable.
  • the microfluidic device can include an acoustic actuator.
  • the loaded sample (biological/chemical material) can be well mixed and separated in a small chamber by acoustic wave before sending out to the nozzle region for encapsulation.
  • the frequency of the acoustic wave should be fine tuned so as not to cause any damage to the cells.
  • the biological effects of acoustic mixing have been well studied (e.g., in the ink-jet industry) and many published literatures also showed that piezoelectric microfluidic device can deliver intact biological payloads such as live microorganisms and DNA.
  • the design of the acoustic resonant can use a Piezoelectric bimorph flat plate located on the side of the carved resonant in the PDMS slab.
  • the resonant inlet can connect to the cell flow input channel and the outlet can connect to the cell flow pinching channel.
  • the piezoelectric driving way form can be carefully optimized to select the critical frequencies that can separate cells in fluids. There are five parameters to optimize beyond the frequency parameter and Lab electronics can be used to optimize the piezoelectric driving waveform. Afterwards, a low cost circuit can be designed to generate only the optimized waveform in a preferred microfluidic device.
  • the microfluidic device of the present invention also includes one or more coalescence modules.
  • a “coalescence module” is within or coincident with at least a portion of the main channel at or downstream of the inlet module where molecules, cells, small molecules or particles comprised within droplets are brought within proximity of other droplets comprising molecules, cells, small molecules or particles and where the droplets in proximity fuse, coalesce or combine their contents.
  • the coalescence module can also include an apparatus, for generating an electric force.
  • the electric force exerted on the fluidic droplet may be large enough to cause the droplet to move within the liquid.
  • the electric force exerted on the fluidic droplet may be used to direct a desired motion of the droplet within the liquid, for example, to or within a channel or a microfluidic channel (e.g., as further described herein), etc.
  • a linker can be used to couple the dye to the bead.
  • the linker can be varied so as to allow the dye to have differing degrees of freedom in which to rotate (i.e., tumble). Varying the linker in this manner can change the FP of the attached dye, which in unique combinations can be used as a label.
  • the beads can be swollen in organic solvent and the dyes held in place by hydrophobic forces.
  • the FP, FI, FL methods described above for liquid labeling can also be used as a means for labeling the beads.
  • a quenching molecule can also be used to change the characteristics of a dye.
  • Such quenching can be continuous or brought about through the interaction of a molecule, such as a peptide or nucleic acid linker, with differing means of bringing molecules together depending on the strength of linker-internal interaction (e.g., a nucleotide stem loop structure of varying lengths).
  • a molecule such as a peptide or nucleic acid linker
  • the reactions analyzed on the virtual, random and non-random arrays can be also increased beyond the two (cy3 and cy5 intensities) commonly used for multiplexing.
  • different FP, FI, etc can be used as a read-out.
  • the invention described herein is superior to the methods of the prior art in that the FP, FI, FL-labeled bead or mobile solid support can be placed into a random array (e.g., a chip as manufactured by Illumina) and the FP, FI, FL used to decode the bead.
  • the FP, FI, FL of the bead can be decoded before using the chip and the different beads ‘mapped’ as to their specific locations.
  • the bead can be decoded during attachment of the assay read-out.
  • the methods described by the present invention can be used to pre-determine the location of each bead-type either before, or during analysis.
  • the detection module may include an apparatus for stimulating a reporter for that characteristic to emit measurable light energy, e.g., a light source such as a laser, laser diode, light emitting diode (LED), high-intensity lamp, (e.g., mercury lamp), and the like.
  • a lamp e.g., the channels are preferably shielded from light in all regions except the detection module.
  • the laser can be set to scan across a set of detection modules from different analysis units.
  • laser diodes or LED's may be microfabricated into the same chip that contains the analysis units.
  • laser diodes or LED's may be incorporated into a second chip (i.e., a laser diode chip) that is placed adjacent to the analysis or microchip such that the laser light from the diodes shines on the detection module(s).
  • the device of the present invention can comprise features, such as integrated metal alloy components and/or features patterned in an electrically conductive layer, for detecting droplets by broadcasting a signal around a droplet and picking up an electrical signal in proximity to the droplet.
  • a fluidic droplet may be directed by creating an electric charge (e.g., as previously described) on the droplet, and steering the droplet using an applied electric field, which may be an AC field, a DC field, etc.
  • an electric field maybe selectively applied and removed (or a different electric field may be applied) as needed to direct the fluidic droplet to a particular region.
  • the electric field may be selectively applied and removed as needed, in some embodiments, without substantially altering the flow of the liquid containing the fluidic droplet.
  • a liquid may flow on a substantially steady-state basis (i.e., the average flowrate of the liquid containing the fluidic droplet deviates by less than 20% or less than 15% of the steady-state flow or the expected value of the flow of liquid with respect to time, and in some cases, the average flowrate may deviate less than 10% or less than 5%) or other predetermined basis through a fluidic system of the invention (e.g., through a channel or a microchannel), and fluidic droplets contained within the liquid may be directed to various regions, e.g., using an electric field, without substantially altering the flow of the liquid through the fluidic system.
  • a substantially steady-state basis i.e., the average flowrate of the liquid containing the fluidic droplet deviates by less than 20% or less than 15% of the steady-state flow or the expected value of the flow of liquid with respect to time, and in some cases, the average flowrate may deviate less than 10% or less than 5%
  • a fluidic system of the invention e.g.
  • the fluidic droplets may be screened or sorted within a fluidic system of the invention by altering the flow of the liquid containing the droplets. For instance, in one set of embodiments, a fluidic droplet may be steered or sorted by directing the liquid surrounding the fluidic droplet into a first channel, a second channel, etc.
  • pressure within a fluidic system can be controlled to direct the flow of fluidic droplets.
  • a droplet can be directed toward a channel junction including multiple options for further direction of flow (e.g., directed toward a branch, or fork, in a channel defining optional downstream flow channels).
  • Pressure within one or more of the optional downstream flow channels can be controlled to direct the droplet selectively into one of the channels, and changes in pressure can be effected on the order of the time required for successive droplets to reach the junction, such that the downstream flow path of each successive droplet can be independently controlled.
  • the expansion and/or contraction of liquid reservoirs may be used to steer or sort a fluidic droplet into a channel, e.g., by causing directed movement of the liquid containing the fluidic droplet.
  • the liquid reservoirs may be positioned such that, when activated, the movement of liquid caused by the activated reservoirs causes the liquid to flow in a preferred direction, carrying the fluidic droplet in that preferred direction.
  • the expansion of a liquid reservoir may cause a flow of liquid towards the reservoir, while the contraction of a liquid reservoir may cause a flow of liquid away from the reservoir.
  • the expansion and/or contraction of the liquid reservoir may be combined with other flow-controlling devices and methods, e.g., as described herein.
  • Non limiting examples of devices able to cause the expansion and/or contraction of a liquid reservoir include pistons and piezoelectric components.
  • piezoelectric components may be particularly useful due to their relatively rapid response times, e.g., in response to an electrical signal.
  • the fluidic droplets may be sorted into more than two channels. Alternately, a fluidic droplet may be sorted and/or split into two or more separate droplets, for example, depending on the particular application. Any of the above-described techniques may be used to spilt and/or sort droplets.
  • a fluidic droplet may be directed to a first region or channel; by applying (or removing) a second electric field to the device (or a portion thereof), the droplet may be directed to a second region or channel; by applying a third electric field to the device (or a portion thereof), the droplet may be directed to a third region or channel; etc., where the electric fields may differ in some way, for example, in intensity, direction, frequency, duration, etc.
  • each droplet may be independently sorted and/or split; for example, some droplets may be directed to one location or another, while other droplets may be split into multiple droplets directed to two or more locations.
  • the present invention provides a device comprising multiple channels with the appropriate geometry to split droplets, perform different experiments on the two daughter droplets and then reorder so that they pass sequential through the detector. The sums, ratios or differences in the two signals can then be calculated before the droplets enter the sorting bifurcation.
  • An indicator dye or equivalent material may be added to one or both droplets to indicate when each droplet enters and leaves the laser.
  • a representative sketch is shown in FIG. 23 .
  • the present invention proposes methods for recovering aqueous phase components from aqueous emulsions that have been collected on a microfluidic device in a minimum number of steps and in a gentle manner so as to minimize potential damage to cell viability.
  • a stable aqueous sample droplet emulsion containing aqueous phase components in a continuous phase carrier fluid is allowed to cream to the top of the continuous phase carrier oil.
  • the continuous phase carrier fluid can include perfluorocarbon oil that can have one or more stabilizing surfactants.
  • the aqueous emulsion rises to the top or separates from the continuous phase carrier fluid by virtue of the density of the continuous phase fluid being greater than that of the aqueous phase emulsion.
  • the perfluorocarbon oil used in one embodiment of the device is 1.8, compared to the density of the aqueous emulsion, which is 1.0.
  • the creamed emulsion is then placed onto a second continuous phase carrier fluid which contains a de-stabilizing surfactant, such as a perfluorinated alcohol (e.g., 1H, 1H, 2H,2H-Perfluoro-1-octanol).
  • a de-stabilizing surfactant such as a perfluorinated alcohol (e.g., 1H, 1H, 2H,2H-Perfluoro-1-octanol).
  • the second continuous phase carrier fluid can also be a perfluorocarbon oil.
  • Additional destabilizing surfactants and/or oil combinations can be identified or synthesized to be useful with this invention.
  • the microfluidic device of the present invention can further include one or more mixing modules.
  • coalescence of one or more droplets in one or more coalescence modules can be sufficient to mix the contents of the coalesced droplets (e.g., through rotating vortexes existing within the droplet), it should be noted that when two droplets fuse or coalesce, perfect mixing within the droplet does not instantaneously occur.
  • the coalesced droplet may initially be formed of a first fluid region (from the first droplet) and a second fluid region (from the second droplet).
  • the fluid regions may remain as separate regions, for example, due to internal “counter-revolutionary” flow within the fluidic droplet, thus resulting in a non-uniform fluidic droplet.
  • a “mixing module” can comprise features for shaking or otherwise manipulate droplets so as to mix their contents.
  • the mixing module is preferably downstream from the coalescing module and upstream from the detection module.
  • the mixing module can include, but is not limited to, the use of channel geometries, acoustic actuators, metal alloy component electrodes or electrically conductive patterned electrodes to mix the contents of droplets and to reduce mixing times for fluids combined into a single droplet in the microfluidic device.
  • the fluidic droplet may be passed through one or more channels or other systems which cause the droplet to change its velocity and/or direction of movement. The change of direction may alter convection patterns within the droplet, causing the fluids to be at least partially mixed. Combinations are also possible.
  • the frequency of the acoustic wave should be fine tuned so as not to cause any damage to the cells.
  • the biological effects of acoustic mixing have been well studied (e.g., in the ink-jet industry) and many published literatures also showed that piezoelectric microfluidic device can deliver intact biological payloads such as live microorganisms and DNA.
  • the design of the acoustic resonant uses a Piezoelectric bimorph flat plate located on the side of the carved resonant in the PDMS slab.
  • the piezoelectric driving waveform is carefully optimized to select the critical frequencies that can separate cells in fluids. There are five parameters to optimize beyond the frequency parameter. Lab electronics is used to optimize the piezoelectric driving waveform. Afterwards, a low cost circuit can be designed to generate only the optimized waveform in a preferred microfluidic device.
  • the microfluidic device of the present invention can further include one or more delay modules.
  • the “delay module” can be a delay line.
  • the operation of a microfluidics device where a reaction within a droplet is allowed to occur for a non-trivial length of time requires a delay line to increase the residence time within the device. For reactions demanding extensive residence time, longer or larger delay lines are required. Accordingly, the invention provides methods to increase residence times within microfluidic devices.
  • the delay module is in fluid communication with the main channel or it can be an elongated portion of the main channel itself.
  • the delay module can be located downstream of the coalescence module and upstream of the detection module.
  • the delay module can be a serpentine channel or a buoyant hourglass.
  • the delay module can further comprise heating and cooling regions. The heating and cooling regions can be used for performing on-chip, flow-through PCR as further described herein.
  • the channel dimensions and configurations can be designed to accommodate the required residence time with minimum pressure drops across the device.
  • the device can comprise a multilayered PDMS slab which is composed of several patterned PDMS slabs.
  • the channel dimensions can also be designed so as to allow for required flow, residence time and pressure drop. Some channels may be required to be very large in width and height.
  • the device includes support posts within the channel design. In order to reduce dead volume behind posts and further improve droplet stability, the support posts are designed to optimize a streamlined flow within the channel. These designs can include curved features as opposed to sharp edges.
  • delay lines can also be extended to the outside of the chip.
  • the off-chip delay lines can be tubes within micron-sized internal diameter.
  • the delay lines can be in the form of a tower (i.e., a structure which is vertical with respect to the ambient gravitational field) as to allow buoyant forces to assist controlled droplet transport.
  • Known delay lines involve transporting droplets by emulsifying them in a carrier fluid flowing in a channel and/or tube. Because the velocity profile of the carrier fluid through the cross-section of the channel and/or tube is not uniform, the velocity distribution of the droplets will not be narrow, which causes the delay time distribution of the droplets to not be narrow (i.e., some droplets will be delayed more or less than others).
  • the devices of the present invention can also include buoyancy-assisted microfluidic delay lines.
  • buoyancy-assisted microfluidic delay lines buoyant forces act on droplets emulsified in a fluid in one or more towers. This can include allowing the tower to fill for the desired delay time, and then releasing the droplets.
  • the tower can or can not continue to fill and release droplets as needed.
  • Droplets that have a density less than their carrier fluid are fed into the base of the tower, buoyantly rise to the top of the tower with a substantially uniform velocity distribution, and are funneled into a functional component of the microfluidic device (such as a y-branch).
  • Carrier fluid is exhausted at the base of the tower at the same rate as it is introduced at the apex so that the net flow of carrier fluid through the delay line is zero.
  • the tower and funnel sections can have any cross-sectional shape, such as circular, elliptical, or polygonal.
  • the microfluidic device can include a tower with adjustable length.
  • the capacity of each tower is 0.05*T, where T is the delay time.
  • the concept includes, for example: (a) upon device start-up, filling the first tower for 0.05*T, but stop-cock its exhaust, and also have the other nineteen towers closed; (b) after 0.05*T, closing the first tower and filling the second between 0.05*T and 0.10*T; (c) repeating step (b) for the remaining eighteen towers; (d) at time T, allowing the first tower to exhaust; (e) at time 1.05*T, stop-cocking the exhaust of the first tower, allowing the second tower to exhaust, and allowing the first tower to fill; (f) at time 1.10*T, stop-cocking the exhaust of the second tower, allowing the third tower to exhaust, closing the first tower, and allowing the second tower to fill, and (g) repeating step (f) a
  • the delay module can also include channels (e.g. the main channel) which has an altered geometry which permits the “parking” (e.g., slowing or stopping) of droplets within the microfluidic device.
  • droplets are able to be parked in wells or channels at predefined locations. This can be done by creating discrete well-like indentions in the channel whereby a droplet ‘falls’ into the well and remains there as the fluid flows over it, or by using a technique entitled ‘by-pass pots’ whereby a droplet is used to block a small outlet in a well, thereby causing the flow to by-pass that droplet-containing well.
  • the instant invention is to use either of these techniques or any related technique, for example just stopping the drops in a channel, to position droplets at either random or predefined places within a microfluidics device. These random or predefined locations can then be queried at a later time-point for a reaction to have occurred, or for removal of the droplets using another means such as resuspension followed by aspiration.
  • a rolling circle amplification reaction is initiated in droplets, the droplets are then parked within the chip, and the amplification reaction allowed to proceed for a set period of time prior to stopping the reaction through the use of heat.
  • the parked droplets are then dried in situ and the covering of the chip disassembled from the chip.
  • One or a set of needle-like devices that are able to be lined up with the droplet parking space are then placed adjacent to or on top of the dried droplets and a liquid solution used to resuspend the material in the dried droplet that has been deposited into the chip, for further downstream processing.
  • the first reactions are created in 10 ⁇ m droplets, the droplets are dried within a channel parking space or by-pass pot which is able to hold a droplet of size larger than 10 ⁇ m, and the droplets are dried in situ.
  • a second set of droplets that are larger than 10 ⁇ m are then allowed to proceed down said channel and when caught in said parking space or by-pass pot are able to resuspend the material from the first droplets that are dried along the walls of the first parking space or by-pass pot.
  • the second droplet is slightly larger than the first and that ensures that the material along the walls is ‘captured’ by the second droplet, and not allowed to diffuse away from the first droplet wall by diffusion.
  • use of surfactants becomes optional in either the first or second droplet formulations.
  • the instant invention also provides the following devices and methods for use in practicing the methods described herein.
  • the PDMS substrate which comprises a portion of the microfluidic device can be covered or coated with an adhesive tape or strip that can removed by peeling.
  • the PDMS substrate can also be bonded by an ultra thin silica that can be pierced by a set of needles.
  • the silica may be spin coated or electro-plated onto a thin backing.
  • Droplets can be dried onto a piece of paper such that can be detected by a second device to determine the Ncode within the droplet and to determine whether an amplification reaction has occurred within the droplet.
  • a plate read comprising dried and undried spots using either an optical array device, such as found in high-end cameras or fiber, optic device is also contemplated.
  • Dry Nitrogen can be utilized to dry the spots by either flowing it through the channel or placing the device into a dry-N2 chamber.
  • Channels can be filled with dried nitrogen or salt run underneath or adjacent to the parking space channels to allow chemical or physical-type gradients to be set up in the chip.
  • the channel walls can be coated with Steptavidin and the produced reactants, for example, DNA biotinylated so that it adheres in situ.
  • Porous beads deposited into the wells can be used in combination with solutions without oils to wash the beads by flow, followed by re-depositing droplets with surfactants to recoat the beads.
  • the wells within the substrate can be filled with many small beads by loading small beads into droplets, storing the droplets into individual wells containing apertures that are slightly smaller than the beads, breaking the droplets by drying or flow of aqueous solutions with or without surfactants into the channels and past the beads, and then re-encapsulating the beads in situ.
  • a set of electrodes within or adjacent to the microfluidic substrate can be used to fuse two droplets in a storage/holding space. The electrodes may be perpendicular to the plane of the channels and either the electrodes or channels moved so as to allow droplet fusions to occur.
  • the present invention also provides methods for chemical synthesis on a bead and releasing said chemical attached to the bead using a releasing means (chemical, UV light, heat, etc.) within a droplet, and then combining a second droplet to the first droplet for further manipulation.
  • a releasing means chemical, UV light, heat, etc.
  • the releasing means is a UV-module.
  • tea-bag synthesis of chemicals on a bead simultaneously with a means for identifying said bead (using, for example, a mass spec tag).
  • a means for identifying said bead using, for example, a mass spec tag.
  • kits also typically include instructions for carrying out the subject assay, and may optionally include the fluid receptacle, e.g., the cuvette, multiwell plate, microfluidic device, etc. in which the reaction is to be carried out.
  • the fluid receptacle e.g., the cuvette, multiwell plate, microfluidic device, etc. in which the reaction is to be carried out.
  • the microfluidic device of the present invention can be utilized to conduct numerous chemical and biological assays, including but not limited to, creating emulsion libraries, flow cytometry, gene amplification, isothermal gene amplification, DNA sequencing, SNP analysis, drug screening, RNAi analysis, karyotyping, creating microbial strains with improved biomass conversion, moving cells using optical tweezer/cell trapping, transformation of cells by electroporation, ⁇ TAS, and DNA hybridization.
  • chemical and biological assays including but not limited to, creating emulsion libraries, flow cytometry, gene amplification, isothermal gene amplification, DNA sequencing, SNP analysis, drug screening, RNAi analysis, karyotyping, creating microbial strains with improved biomass conversion, moving cells using optical tweezer/cell trapping, transformation of cells by electroporation, ⁇ TAS, and DNA hybridization.
  • “about” or “approximately” shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range.
  • molecule means any distinct or distinguishable structural unit of matter comprising one or more atoms, and includes for example polypeptides and polynucleotides.
  • polymer means any substance or compound that is composed of two or more building blocks (‘mers’) that are repetitively linked to each other.
  • a “dimer” is a compound in which two building blocks have been joined together.
  • polynucleotide refers to a polymeric molecule having a backbone that supports bases capable of hydrogen bonding to typical polynucleotides, where the polymer backbone presents the bases in a manner to permit such hydrogen bonding in a sequence specific fashion between the polymeric molecule and a typical polynucleotide (e.g., single-stranded DNA).
  • bases are typically inosine, adenosine, guanosine, cytosine, uracil and thymidine.
  • Polymeric molecules include double and single stranded RNA and DNA, and backbone modifications thereof, for example, methylphosphonate linkages.
  • nucleotide sequence is a series of nucleotide bases (also called “nucleotides”) generally in DNA and RNA, and means any chain of two or more nucleotides.
  • a nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double or single stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and anti-sense polynucleotide (although only sense stands are being represented herein).
  • PNA protein nucleic acids
  • polynucleotides herein may be flanked by natural regulatory sequences, or may be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5′- and 3 ′-non-coding regions, and the like.
  • the nucleic acids may also be modified by many means known in the art.
  • Non-limiting examples of such modifications include methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.
  • charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • Polynucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators.
  • the polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage.
  • the polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
  • DNA deoxyribonucleic acid
  • DNA means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and thymine (T), called nucleotide bases, that are linked together on a deoxyribose sugar backbone.
  • DNA can have one strand of nucleotide bases, or two complimentary strands which may form a double helix structure.
  • RNA ribonucleic acid
  • RNA ribonucleic acid
  • RNA typically has one strand of nucleotide bases.
  • a “polypeptide” (one or more peptides) is a chain of chemical building blocks called amino acids that are linked together by chemical bonds called peptide bonds.
  • a “protein” is a polypeptide produced by a living organism.
  • a protein or polypeptide may be “native” or “wild-type”, meaning that it occurs in nature; or it may be a “mutant”, “variant” or “modified”, meaning that it has been made, altered, derived, or is in some way different or changed from a native protein, or from another mutant.
  • particles means any substance that may be encapsulated within a droplet for analysis, reaction, sorting, or any operation according to the invention.
  • Particles are not only objects such as microscopic beads (e.g., chromatographic and fluorescent beads), latex, glass, silica or paramagnetic beads, but also includes other encapsulating porous and/or biomaterials such as liposomes, vesicles and other emulsions. Beads ranging in size from 0.1 micron to 1 mm can be used in the devices and methods of the invention and are therefore encompassed with the term “particle” as used herein.
  • the term particle also encompasses biological cells, as well as beads and other microscopic objects of similar size (e.g., from about 0.1 to 120 microns, and typically from about 1 to 50 microns) or smaller (e.g., from about 0.1 to 150 nm).
  • the devices and methods of the invention are also directed to sorting and/or analyzing molecules of any kind, including polynucleotides, polypeptides and proteins (including enzymes) and their substrates and small molecules (organic or inorganic).
  • the term particle further encompasses these materials.
  • the particles are sorted and/or analyzed by encapsulating the particles into individual droplets (e.g., droplets of aqueous solution in oil), and these droplets are then sorted, combined and/or analyzed in a microfabricated device.
  • droplets generally includes anything that is or can be contained within a droplet.
  • a “small molecule” as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD.
  • Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art.
  • cell means any cell or cells, as well as viruses or any other particles having a microscopic size, e.g. a size that is similar to or smaller than that of a biological cell, and includes any prokaryotic or eukaryotic cell, e.g., bacteria, fungi, plant and animal cells.
  • Cells are typically spherical, but can also be elongated, flattened, deformable and asymmetrical, i.e., non-spherical.
  • the size or diameter of a cell typically ranges from about 0.1 to 120 microns, and typically is from about 1 to 50 microns.
  • a cell may be living or dead.
  • the microfabricated device of the invention is directed to sorting materials having a size similar to a biological cell (e.g. about 0.1 to 120 microns) or smaller (e.g., about 0.1 to 150 nm) any material having a size similar to or smaller than a biological cell can be characterized and sorted using the microfabricated device of the invention.
  • the term cell shall further include microscopic beads (such as chromatographic and fluorescent beads), liposomes, emulsions, or any other encapsulating biomaterials and porous materials.
  • Non-limiting examples include latex, glass, orparamagnetic beads; and vesicles such as emulsions and liposomes, and other porous materials such as silica beads.
  • Beads ranging in size from 0.1 micron to 1 mm can also be used, for example in sorting a library of compounds produced by combinatorial chemistry.
  • a cell may be charged or uncharged.
  • charged beads may be used to facilitate flow or detection, or as a reporter.
  • Biological cells, living or dead may be charged for example by using a surfactant, such as SDS (sodium dodecyl sulfate).
  • SDS sodium dodecyl sulfate
  • the term cell further encompasses “virions”, whether or not virions are expressly mentioned.
  • a “virion”, “virus particle” is the complete particle of a virus.
  • Viruses typically comprise a nucleic acid core (comprising DNA or RNA) and, in certain viruses, a protein coat or “capsid” Certain viruses may have an outer protein covering called an “envelope”.
  • a virion may be either living (i.e., “viable”) or dead (i.e., “non-viable”).
  • a living or “viable” virus is one capable of infecting a living cell.
  • Viruses are generally smaller than biological cells and typically range in size from about 20-25 nm diameter or less (parvoviridae, picornoviridae) to approximately 200-450 nm (poxviridae).
  • filamentous viruses may reach lengths of 2000 nm (closterviruses) and are therefore larger than some bacterial cells.
  • the microfabricated device of the invention is particularly suited for sorting materials having a size similar to a virus (i.e., about 0.1 to 150 nm)
  • any material having a size similar to a virion can be characterized and sorted using the microfabricated device of the invention.
  • Non-limiting examples include latex, glass or paramagnetic beads; vesicles such as emulsions and liposomes; and other porous materials such as silica beads. Beads ranging in size from 0.1 to 150 nm can also be used, for example, in sorting a library of compounds produced by combinatorial chemistry.
  • a virion may be charged or uncharged.
  • charged beads may be used to facilitate flow or detection, or as a reporter.
  • Biological viruses whether viable or non-viable, may be charged, for example, by using a surfactant, such as SDS.
  • a “reporter” is any molecule, or a portion thereof, that is detectable, or measurable, for example, by optical detection.
  • the reporter associates with a molecule, cell or virion or with a particular marker or characteristic of the molecule, cell or virion, or is itself detectable to permit identification of the molecule, cell or virion's, or the presence or absence of a characteristic of the molecule, cell or virion.
  • characteristics include size, molecular weight, the presence or absence of particular constituents or moieties (such as particular nucleotide sequences or restrictions sites).
  • reporter In the case of cells, characteristics which may be marked by a reporter includes antibodies, proteins and sugar moieties, receptors, polynucleotides, and fragments thereof.
  • label can be used interchangeably with “reporter”.
  • the reporter is typically a dye, fluorescent, ultraviolet, or chemiluminescent agent, chromophore, or radio-label, any of which may be detected with or without some kind of stimulatory event, e.g., fluoresce with or without a reagent.
  • the reporter is a protein that is optically detectable without a device, e.g. a laser, to stimulate the reporter, such as horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • a protein reporter can be expressed in the cell that is to be detected, and such expression may be indicative of the presence of the protein or it can indicate the presence of another protein that may or may not be coexpressed with the reporter.
  • a reporter may also include any substance on or in a cell that causes a detectable reaction, for example by acting as a starting material, reactant or a catalyst for a reaction which produces a detectable product. Cells may be sorted, for example, based on the presence of the substance, or on the ability of the cell to produce the detectable product when the reporter substance is provided.
  • a “marker” is a characteristic of a molecule, cell or virion that is detectable or is made detectable by a reporter, or which may be coexpressed with a reporter.
  • a marker can be particular constituents or moieties, such as restrictions sites or particular nucleic acid sequences in the case of polynucleotides.
  • characteristics may include a protein, including enzyme, receptor and ligand proteins, saccharrides, polynucleotides, and combinations thereof, or any biological material associated with a cell or virion.
  • the product of an ‘enzymatic reaction may also be used as a marker.
  • the marker may be directly or indirectly associated with the reporter or can itself be a reporter.
  • a marker is generally a distinguishing feature of a molecule, cell or virion
  • a reporter is generally an agent which directly or indirectly identifies or permits measurement of a marker.
  • the present invention provides methods for preparing a library of droplet emulsions, where each of the droplets is of the same, predetermined size (monodisperse). Further, present invention provides methods for deterministic lateral displacement for continuous particle separation, which can occur within droplets on a microfluidic device.
  • Particles in solution are usually separated according to size by exclusion or hydrodynamic chromatography.
  • a sample mixture is injected at one end of a tube packed with porous beads and then washed through the tube. Particles smaller than the pore sizes enter the beads, which lengthen their migration path, and so they are on average eluted later than larger particles. Zones of particles of a given size broaden, however, because particles in each zone take many different paths leading to different retention times. This multipath effect reduces the resolution of size-exclusion chromatography.
  • hydrodynamic chromatography a sample mixture is driven through a capillary by hydrodynamic flow, which has a parabolic flow profile. Large particles cannot intercept the low-velocity fluid near the capillary wall, and thus on average move faster and become separated from small particles. Multipath effects also limit the resolution of hydrodynamic chromatography, because each migration path samples different velocities in the parabolic flow.
  • Lateral displacement means for sizing and separating droplets in solution can be utilized.
  • the present invention relates to the generation of a microfluidic device consisting of raised pillars in both columns and rows that are designed for lateral diffusion.
  • the pillars can be adjusted so as to be a means for separating droplets of similar sizes from a fluid containing various sized droplets.
  • a fluid containing oil, water and a surfactant is mixed so as to create a bulk emulsion.
  • the bulk emulsion is injected into beginning of a microfluidic lateral diffusion device and various fractions are collected at the ending of the device at positions corresponding to specific sizes.
  • Advantages to this lateral diffusion separation means would be the isolation of similarly-sized droplets off-line in a fast and facile manner.
  • Bulk emulsions could be size-selected and then the resulting emulsions, if desired, combined to create sized libraries for re-introduction into a microfluidic device.
  • the lateral diffusion microfluidic devices could be rolled-up into a syringe or designed for parallel processing.
  • Microfabricated devices have also been designed that inherently rely on diffusion for separation. Particle mixtures are either repeatedly subject to spatially asymmetric potentials created by microelectrodes or driven through arrays of micrometer-scale asymmetric obstacles to exploit differences in diffusion lengths. In all of the devices discussed so far, particles in a given zone have many different migration paths, and diffusion is required for separation.
  • the present invention describes a separation process that creates equivalent migration paths for each particle in a mixture, thereby eliminating multipath zone broadening ( FIG. 24 ).
  • FIG. 24 (Panel A) Geometric parameters defining the obstacle matrix. A fluid flow is applied in the vertical direction (orange arrow).
  • Panel C A particle with a radius that is larger than lane 1 follows a streamline passing through the particle's center (black dot), moving toward lane 1. The particle is physically displaced as it enters the next gap. Black dotted lines mark the lanes.
  • the separation process uses laminar flow through a periodic array of micrometer-scale obstacles. Each row of obstacles is shifted horizontally with respect to the previous row by ⁇ , where ⁇ is the center-to-center distance between the obstacles ( FIG. 24 ). For convenience, let ⁇ / ⁇ be 1 ⁇ 3. Fluid emerging from a gap between two obstacles will encounter an obstacle in the next row and will bifurcate as it moves around the obstacle. Let the flow diverted to the left of the obstacle be ⁇ , where ⁇ is the total fluid flux going through the gap. If the fluid is confined to move straight down through the array, ⁇ must equal ⁇ / ⁇ . Let us then consider the flow through a gap to be made up of three lanes, each of which by definition has a flux of ⁇ /3.
  • Particles that are smaller than the lane width will follow the streamlines.
  • a particle starting in lane 1 will go through lane 3 (right lane with respect to the gap) in the second row, lane 2 (middle lane) in the third row, and back to lane 1 (left lane) in the fourth row ( FIG. 24 B ).
  • particles starting from any of the three lanes will go back to the original lane assignment after three rows, so that net migration is in the average flow direction. This motion is called the “zigzag mode.”
  • particles can diffuse into an adjacent lane.
  • the microscopic path for all lanes is equivalent, unlike the multiple paths particles take when moving through a column of porous beads.
  • FIG. 26 is a schematic illustrating the separation by deterministic lateral displacement in an array of microposts, with an example row shift fraction of one-third. This shift creates three equal flux streamlines.
  • the dashed lines are the boundaries between the streamlines, which are assigned an index in the gaps between the posts.
  • Paths of particles both smaller and larger than the critical threshold are depicted with green and red dotted lines respectively. Small particles stay within a flow Stream and large particles are displaced at each obstacle.
  • G is the clear spacing between the gap, is the center-to-center post separation, and d is the relative shift of the post centers in adjacent rows.
  • a PCR primer can be synthesized with a universal sequencing primer binding site added to the 5′ end (e.g., see Appendix E in Applied Biosystems' “Automated DNA Sequencing: Chemistry Guide” for universal primer sequences). This allows any PCR product to be sequenced with universal primers.
  • Universal-tailed PCR primers enable the use of commercially available dye-labeled sequencing primers. This technique is also useful with dye terminator chemistries, because universal sequencing primers have good annealing characteristics. However, the longer PCR primers add to the overall cost of the reactions. Using universal-tailed primers sometimes results in primer oligomerization. As these products have priming sites present, they can result in noisy data for the first 20-100 bases. Redesigning the PCR primer, optimizing the PCR amplification further, and employing Hot Start methods can help overcome this situation.
  • SAP/Exo I shrimp alkaline phosphatase
  • Exo I exonuclease I
  • the SAP/Exo I procedure degrades nucleotides and single-stranded DNA (primers) remaining after PCR (Werle et al., 1994). This procedure is particularly useful in cases where limiting concentrations of primers and nucleotides cannot be used for direct PCR sequencing.
  • FIG. 27 shows one embodiment for a DNA sequencing chip design.
  • Template DNA and primers are combined at step ‘add 1’ and the reaction is incubated at 95° C. for a hot start (position 1).
  • the reaction then cycles 20-30 times (position 2) before the addition of SAP and ExoI at ‘add 2.’
  • the reaction is incubated at 37° C. for a pre-defined time-period and then the SAP and Exol enzymes are inactivated at 95° C. (position ‘4’).
  • the SAP/ExoI procedure degrades nucleotides and single-stranded DNA (primers) remaining after PCR.
  • the universal sequencing primers, ddNTPs and buffers are added at ‘add 3,’ and the PCR sequencing reaction is allowed to proceed at position ‘5.’
  • the final reaction product is collected and can be stored off-chip.
  • PCR protocols that limit amounts of primers and dNTPs allow the product of the reaction to be used for sequencing with no purification. This is usually carried out by setting up the PCR amplification with 5-10 pmol of primers and 20-40 ⁇ M dNTPs, so that most of the primers and dNTPs are exhausted during amplification. If the yield of the desired PCR product is high and the product is specific, i.e., it produces a single band when analyzed by agarose gel electrophoresis, the sample can be diluted before sequencing and will give good results. The dilution ratio depends on the concentration of your PCR product and needs to be determined empirically (start with 1:2 and 1:10 dilutions with deionized water).
  • Direct PCR sequencing is most useful in applications where the same target is being amplified and sequenced repeatedly and PCR conditions have been optimized. Direct PCR sequencing can be done with dye primer chemistries. With dye terminator chemistries, it is much more critical that the PCR primers be consumed. Excess PCR primers will be extended and labeled by the cycle sequencing reaction, resulting in noisy data. Direct PCR sequencing does not work for XL PCR because limiting amounts of primers and dNTPs cannot be used. The PCR product should be purified or the excess primers and nucleotides should be degraded by SAP/Exo I treatment.
  • the present invention provides methods for performing isothermal-type amplification methods on a microfluidic device.
  • Isothermal amplification is an alternative to the standard PCR techniques described herein. Isothermal amplification is used to reduce the relative amount of background DNA in a sample. Primers are generally used in a constant temperature means of amplification. Isothermal amplification is applicable for SNP detection.
  • Once the DNA is amplified by isothermal amplification there are several well-known means for detecting which nucleotide polymorphism is present. These include, but are not limited to; allele specific primer extension, oligonucleotide ligation assay, mini-sequencing, fluorescence polarization, etc.
  • Isothermal amplification is also applicable for DNA sequencing preparation.
  • the isothermally-amplified DNA can be attached to a solid phase within a droplet or placed within a parking space on chip. The beads or parking spaces can be accessed and the amplified DNA used for a DNA sequencing reaction.
  • isothermal amplification is applicable for gene expression analysis. Isothermal amplification can be used to monitor gene expression by the measurement of the amount of cDNA produced in a quantitative fashion. Many methods for isothermal amplification are known in the art, including but not limited to the following examples.
  • Rolling circle amplification A DNA polymerase extends a primer on a circular template, generating tandemly linked copies of the complementary sequence of the template (Fire & Xu, 1995).
  • the TempliPhi amplification process using rolling circle amplification is known in the art. In the process, random hexamer primers anneal to the circular template DNA at multiple sites. Phi29 DNA polymerase extends each of these primers. When the DNA polymerase reaches a downstream-extended primer, strand displacement synthesis occurs. The displaced strand is rendered single-stranded and available to be primed by more hexamer primer. The process continues, resulting in exponential, isothermal amplification.
  • TMA Transcription mediated amplification
  • An RNA polymerase is used to make RNA from a promoter engineered in the primer region, a reverse transcriptase to produce complementary DNA from the RNA templates and RNase H to remove the RNA from cDNA (Guatelli et al, 1990).
  • Strand-displacement amplification SDA
  • a restriction endonuclease is used to nick the unmodified strand of its target DNA and the action of an exonuclease-deficient DNA polymerase to extend the 30 end at the nick and displace the downstream DNA strand (Walker et al, 1992). Strand-displacement amplification is known in the art.
  • HAD Helicase-dependent amplification
  • a DNA helicase is used to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. Schematic diagram of HAD is shown in FIG. 28 . Two complementary DNA strands are shown as two lines: the thick one is the top strand and the thin one is the bottom strand. 1: A helicase (black 30 triangle) separates the two complementary DNA strands, which are bound by SSB (grey circles). 2: Primers (lines with arrow heads) hybridize to the target region on the ssDNA template. 3: A DNA polymerase (squares with mosaic patterns) extends the primers hybridized on the template DNA. 4: Amplified products enter the next round of amplification.
  • Random libraries of DNA fragments are generated by shearing an entire genome and isolating single DNA molecules by limiting dilution. See, FIG. 29 .
  • sequencing reactions such as those performed by Solexa, 454 Life Sciences and others involve randomly fragmenting the entire genome, adding specialized common adapters to the fragments, capturing the individual fragments on their own beads and, within the droplets of an emulsion, clonally amplifying the individual fragment ( FIG. 29 a , 29 b ).
  • their approach does not require subcloning or the handling of individual clones; the templates are handled in bulk within the emulsions. Typically, about 30% of the beads will have DNA, producing 450,000 template-carrying beads per emulsion reaction.
  • FIG. 29 Sample preparation and DNA sequencing is shown in FIG. 29 .
  • Panel A Genomic DNA is isolated, fragmented, ligated to adapters and separated into single strands (top left). Fragments are bound to beads under conditions that favor one fragment per bead, the beads are captured in the droplets of a PCR-reaction-mixture-in-oil emulsion and PCR amplification occurs within each droplet, resulting in beads each carrying ten million copies of a unique DNA template (top, second from the left). The emulsion is broken, the DNA strands are denatured, and beads carrying single-stranded DNA clones are deposited into wells of a fiber-optic slide (bottom left).
  • Panel B Microscope photograph of emulsion showing droplets containing a bead and empty droplets. The thin arrow points to a 28-mm bead; the thick arrow points to an approximately 100-mm droplet.
  • Panel C Scanning electron micrograph of a portion of a fiber-optic slide, showing fiber-optic cladding and wells before bead deposition.
  • Panel D The sequencing instrument consists of the following major subsystems: a fluidic assembly.
  • Panel E a flow chamber that includes the well-containing fiber-optic slide.
  • Panel F a CCD camera-based imaging assembly.
  • Panel G and a computer that provides the necessary user interface and instrument control.
  • Another example is sequencing in fabricated picolitre-sized reaction vessels.
  • One method uses sequencing by synthesis simultaneously in open wells of a fiber-optic slide using a modified pyrosequencing protocol that is designed to take advantage of the small scale of the wells.
  • the fiber optic slides are manufactured by slicing of a fiber-optic block that is obtained by repeated drawing and fusing of optic fibers. At each iteration, the diameters of the individual fibers decrease as they are hexagonally packed into bundles of increasing cross-sectional sizes.
  • Each fiber-optic core is 44 ⁇ m in diameter and surrounded by 2-3 ⁇ m of cladding; etching of each core creates reaction wells approximately 55 ⁇ m in depth with a centre-to-centre distance of 50 ⁇ m ( FIG.
  • the slide containing approximately 1.6 million wells, is loaded with beads and mounted in a flow chamber designed to create a 300- ⁇ m high channel, above the well openings, through which the sequencing reagents flow ( FIG. 29 d ).
  • the unetched base of the slide is in optical contact with a second fiber optic imaging bundle bonded to a charge-coupled device (CCD) sensor, allowing the capture of emitted photons from the bottom of each individual well ( FIG. 29 d ).
  • CCD charge-coupled device
  • reagents flow perpendicularly to the wells.
  • This configuration allows simultaneous extension reactions on template-carrying beads within the open wells and relies on convective and diffusive transport to control the addition or removal of reagents and by-products.
  • the timescale for diffusion into and out of the wells is on the order of s in the current configuration and is dependent on well depth and flow channel height.
  • the timescales for the signal-generating enzymatic reactions are on the order of 0.02-1.5 s.
  • the current reaction is dominated by mass transport effects, and improvements based on faster delivery of reagents are possible.
  • Well depth was selected on the basis of a number of competing requirements: (1) wells need to be deep enough for the DNA-carrying beads to remain in the wells in the presence of convective transport past the wells; (2) they must be sufficiently deep to provide adequate isolation against diffusion of by-products from a well in which incorporation is taking place to a well where no incorporation is occurring; and (3) they must be shallow enough to allow rapid diffusion of nucleotides into the wells and rapid washing out of remaining nucleotides at the end of each flow cycle to enable high sequencing throughput and reduced reagent use. After the flow of each nucleotide, a wash containing a pyrase is used to ensure that nucleotides do not remain in any well before the next nucleotide being introduced.
  • Nucleotide incorporation is detected by the associated release of inorganic pyrophosphate and the generation of photons.
  • Wells containing template-carrying beads are identified by detecting a known four-nucleotide ‘key’ sequence at the beginning of the read.
  • Raw signals are background-subtracted, normalized and corrected.
  • the normalized signal intensity at each nucleotide flow, for a particular well indicates the number of nucleotides, if any, that were incorporated. This linearity in signal is preserved to at least homopolymers of length eight.
  • sequencing by synthesis a very small number of templates on each bead lose synchronism (that is, either get ahead of, or fall behind, all other templates in sequence).
  • NTA Non-template amplification
  • the present invention provides the use of mixed modified and standard hexamer primers in microfluidic reactions to retard NTA while allowing template-based amplification to proceed.
  • the modified primers of the present invention containing nitroindoles and C3 non-replicable elements were studied in an effort to reduce NTA both in bulk and microfluidic reactions. Both nitroindoles and C3 non-replicable elements were found to be effective in reducing NTA, with primers containing two 5′ nitroindoles most effective in NTA suppression. However, increased NTA suppression was tightly linked with reduced yield in template amplification reactions.
  • Amplifications using a ratio of nitroindole to random hexamer primers generated a range of both template and non-template amplification yields, with a 15:85 ratio of nitroindole to random hexamers generating template yields commensurate with random hexamers primers alone, but generating little if any spurious product in the absence of template.
  • a Single Nucleotide Polymorphism is a small genetic change, or variation, that can occur within a person's DNA sequence.
  • the genetic code is specified by the four nucleotide “letters” A (adenine), C (cytosine), T (thymine), and G (guanine).
  • SNP variation occurs when a single nucleotide, such as an A, replaces one of the other three nucleotide letters—C, G, or T.
  • SNP SNP
  • AAGGTTA ATGGTTA
  • T ATGGTTA
  • SNPs SNPs occur in the human population more than 0.1 percent of the time. Because only about 3 to 5 percent of a person's DNA sequence codes for the production of proteins, most SNPs are found outside of “coding sequences”. SNPs found within a coding sequence are of particular interest to researchers because they are more likely to alter the biological function of a protein. Because of the recent advances in technology, coupled with the unique ability of these genetic variations to facilitate gene identification, there has been a recent flurry of SNP discovery and detection.

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Abstract

The present invention provides novel microfluidic substrates and methods that are useful for performing biological, chemical and diagnostic assays. The substrates can include a plurality of electrically addressable, channel bearing fluidic modules integrally arranged such that a continuous channel is provided for flow of immiscible fluids.

Description

RELATED APPLICATIONS
This patent application is a continuation of U.S. Nonprovisional application Ser. No. 13/779,943, filed Feb. 28, 2013, which is a continuation application of U.S. Nonprovisional application Ser. No. 11/803,104, filed May 11, 2007, which claims priority to, and the benefit of, U.S. Provisional Application Nos. 60/799,833 filed on May 11, 2006; 60/799,834 filed on May 11, 2006; 60/808,614 filed on May 25, 2006; 60/815,097 filed on Jun. 19, 2006; 60/819,733 filed on Jul. 7, 2006; 60/819,734 filed on Jul. 7, 2006; 60/841,716 filed on Sep. 1, 2006; 60/843,374 filed on Sep. 8, 2006; 60/833,151 filed on Jul. 24, 2006; 60/834,987 filed on Jul. 31, 2006; 60/837,871 filed on Aug. 14, 2006; 60/837,695 filed on Aug. 14, 2006; 60/843,327 filed on Sep. 8, 2006; 60/856,540 filed on Nov. 3, 2006; 60/856,440 filed on Nov. 3, 2006; 60/874,561 filed on Dec. 12, 2006; 60/858,279 filed on Nov. 8, 2006; 60/858,278 filed on Nov. 8, 2006; 60/874,640 filed on Dec. 12, 2006; 60/860,665 filed on Nov. 22, 2006; 60/873,766 filed on Dec. 8, 2006; 60/876,209 filed on Dec. 20, 2006; 60/899,258 filed on Feb. 2, 2007; 60/903,153 filed on Feb. 23, 2007; 60/904,293 filed on Feb. 28, 2007; 60/920,337 filed on Mar. 26, 2007. The contents of each of these applications are incorporated herein by reference in their entirety.
FIELD OF INVENTION
The present invention generally relates to systems and methods for the formation and/or control of fluidic species, and articles produced by such systems and methods. More particularly, the present invention relates to the development of high throughput microfluidic devices for precision fluid handling and use of such systems in various biological, chemical, or diagnostic assays.
BACKGROUND
The manipulation of fluids to form fluid streams of desired configuration, discontinuous fluid streams, droplets, particles, dispersions, etc., for purposes of fluid delivery, product manufacture, analysis, and the like, is a relatively well-studied art. For example, highly monodisperse gas bubbles, less than 100 microns in diameter, have been produced using a technique referred to as capillary flow focusing. In this technique, gas is forced out of a capillary tube into a bath of liquid, where the tube is positioned above a small orifice, and the contraction flow of the external liquid through this orifice focuses the gas into a thin jet which subsequently breaks into equal-sized bubbles via a capillary instability. A similar arrangement can be used to produce liquid droplets in air.
Microfluidic systems have been described in a variety of contexts, typically in the context of miniaturized laboratory (e.g., clinical) analysis. Other uses have been described as well. For example, International Patent Application Publication No. WO 01/89788 describes multi-level microfluidic systems that can be used to provide patterns of materials, such as biological materials and cells, on surfaces. Other publications describe microfluidic systems including valves, switches, and other components.
Precision manipulation of streams of fluids with microfluidic devices is revolutionizing many fluid-based technologies. Networks of small channels are a flexible platform for the precision manipulation of small amounts of fluids. The utility of such microfluidic devices depends critically on enabling technologies such as the microfluidic peristaltic pump, electrokinetic pumping, dielectrophoretic pump or electrowetting driven flow. The assembly of such modules into complete systems provides a convenient and robust way to construct microfluidic devices. However, virtually all microfluidic devices are based on flows of streams of fluids; this sets a limit on the smallest volume of reagent that can effectively be used because of the contaminating effects of diffusion and surface adsorption. As the dimensions of small volumes shrink, diffusion becomes the dominant mechanism for mixing leading to dispersion of reactants; moreover, surface adsorption of reactants, while small, can be highly detrimental when the concentrations are low and volumes are small. As a result current microfluidic technologies cannot be reliably used for applications involving minute quantities of reagent; for example, bioassays on single cells or library searches involving single beads are not easily performed. An alternate approach that overcomes these limitations is the use of aqueous droplets in an immiscible carrier fluid; these provide a well defined, encapsulated microenvironment that eliminates cross contamination or changes in concentration due to diffusion or surface interactions. Droplets provide the ideal microcapsule that can isolate reactive materials, cells, or small particles for further manipulation and study. However, essentially all enabling technology for microfluidic systems developed thus far has focused on single phase fluid flow and there are few equivalent active means to manipulate droplets requiring the development of droplet handling technology. While significant advances have been made in dynamics at the macro- or microfluidic scale, improved techniques and the results of these techniques are still needed. For example, as the scale of these reactors shrinks, contamination effects due to surface adsorption and diffusion limit the smallest quantities that can be used. Confinement of reagents in droplets in an immiscible carrier fluid overcomes these limitations, but demands new fluid-handling technology.
Furthermore, the underlying physics of the influence of electric fields on fluids is well known. The attractive and repulsive forces produced by an electric field on positive or negative charges give rise to the forces on charged fluid elements, the polarization of non-polar molecules, and the torque on polar molecules which aligns them with the field. In a non-uniform field, because the force on the positively charged portion of the distribution is different than the force on the negatively charged portion, polar molecules will also experience a net force toward the region of higher field intensity. In the continuum limit, the result is a pondermotive force in the fluid. In the limit of high droplet surface tension, it is useful to describe the net pondermotive force on a droplet as if it were a rigid sphere:
F=qE+2π
Figure US12337287-20250624-P00001
(εm)r 3
Figure US12337287-20250624-P00001
(K)∇E 2,
where the first term is the electrophoretic force on the droplet (q is the net droplet charge and E is the electric field), and the second term is the dielectrophoretic force (r is the radius of the sphere,
Figure US12337287-20250624-P00002
(K) is the real part of the Clausius-Mossotti factor
K=(ε*p−ε*m)/(ε*p+2ε*m),
and ε*p and ε*m are the complex permittivities of the droplet and carrier fluid).
Although utility of electrophoretic control of droplets is great, it does have significant limitations. First, the charging of droplets is only effectively accomplished at the nozzle. Second, the discharge path required to eliminate screening effects also discharges the droplets. Third, finite conductivity of the carrier fluid, however small, will eventually discharge the droplets. Therefore, once the droplet is formed, there is essentially only one opportunity to perform any pondermotive function which relies on the droplet's charge density (such as coalescing oppositely charged droplets through their mutual Coulombic attraction, or electrophoretically sorting a droplet), and that function can only be performed as long as sufficient charge has not leaked off of the droplet.
Thus, it would be desirable to develop an electrically addressable emulsification system that combines compartmentalization and electrical manipulation, which allows for multi-step chemical processing, including analysis and sorting, to be initiated in confinement with exquisite timing and metering precision, for use in a variety of chemical, biological, and screening assays, in which the cost and time to perform such assays would be drastically reduced. It would also be desirable to develop a device using dielectrophoretic force (which does not rely on charge density) to manipulate droplets so that more than one electrical pondermotive function can be carried out following a significantly long delay from droplet formation.
SUMMARY OF THE INVENTION
The present invention provides substrates having individual fluid handling modules that can be combined into fluid processing systems so as to perform multi-step processing of isolated components, which is essential to perform biological, chemical and diagnostic applications, quickly, effectively and inexpensively. Using principles based on the electrical manipulation of droplets, the microfluidic substrates of the present invention can encapsulate reagents into droplets, which can be combined, analyzed, and sorted.
The present invention provides a microfluidic substrate. The substrate can include a plurality of Microfluidic modules integrally arranged with each other so as to be in fluid communication. The substrate can include, for example, (i) at least one inlet module having at least one inlet channel adapted to carry at least one dispersed phase fluid, (ii) at least one main channel adapted to carry at least one continuous phase fluid, wherein the inlet channel is in fluid communication with the main channel at a junction, wherein the junction includes a fluidic nozzle designed for flow focusing such that the dispersed phase fluid is immiscible with the continuous phase fluid and forms a plurality of highly uniform, monodisperse droplets in the continuous phase fluid. The flow of the dispersed phase and continuous phase can be pressure driven, for example. The dispersed phase (e.g. droplets) can be neutral or have no charge and these droplets can be manipulated (e.g., coalesced, sorted) within a electric field in the continuous phase fluid.
The inlet module can further include at least one self-aligning fluidic interconnect apparatus to connect the inlet channel to a means for introducing a sample fluid to the channel, wherein the apparatus forms a radial seal between the microfluidic substrate and the means for introducing sample. The means can include, for example, a well or reservoir, which can be temperature controlled. The well or reservoir can optionally include an acoustic actuator.
The microfluidic substrate can include one or more additional modules, including but not limited to, coalescence module, detection module, sorting module, collection module, waste module, delay module (e.g., heating and cooling modules), droplet spacing module, mixing module, UV-release module, division module and/or reordering module. These modules are in fluid communication with the main channel. There may be zero, one, or more of each of the modules.
The substrate can further include at least one coalescence module downstream from and in fluid communication with the inlet module via the main channel including a coalescence apparatus, wherein two or more droplets passing there through are coalesced to form a nanoreactor.
The substrate can further include at least one detection module downstream from and in fluid communication with the coalescence module. The detection module can include, for example, a detection apparatus for evaluating the contents and/or characteristics of the nanoreactor. The detection apparatus can include an optical or electrical detector.
The substrate can further include a sorting module downstream from and in fluid communication with the detection module. The sorting module can include, for example, a sorting apparatus adapted to direct the nanoreactor into or away from a collection module in response to the contents or characterization of the nanoreactor evaluated in the detection module. The channels in the sorting module can include an asymmetric bifurcation geometry or an asymmetric bifurcation flow.
The coalescence apparatus and the sorting apparatus can include one or more electrodes, or a patterned electrically conductive layer, which are capable of generating an electric field. The electrodes can be made from electrically conductive materials, and can be integrally contained in one or more channels isolated from the main and inlet channels of the substrate. The electrically conductive materials can be metal alloy components or organic materials. The electrically conductive material can be an epoxy resin including one or more electrically conductive particles. The electrically conductive particles can be silver particles.
The coalescence module can further include an expanded portion of the main channel between the electrodes to bring successive droplets into proximity, whereby the paired droplets are coalesced within the electric field. The coalescence module can further include a narrowed portion of the main channel to center droplets within the main channel prior to the expanded portion of the main channel between the electrodes.
The channels of the microfluidic substrate can be coated with an anti-wetting or blocking agent for the dispersed phase. The anti-wetting or blocking agent can include, for example, a silica primer layer followed by a perfluoroalkylalkylsilane compound, an amorphous soluble perfloropolymer, BSA, PEG-silane or fluorosilane. The channels of the microfluidic substrate can include well-like indentations to slow, stop or react contents of droplets.
The substrate can further include a collection module connected to a means for storing a sample from the substrate and a waste module connected to a means for collecting a sample discarded from the substrate. The means can be a well or reservoir, which can be temperature controlled.
The substrate can further include a delay module in fluid communication with the main channel downstream of the coalescence module and upstream of the detection module. The delay module can be a delay line, serpentine channel, a buoyant hourglass, or an off-chip volume. Preferably, a serpentine channel is used to time delays less than 1 hour. Preferably, an off-chip volume is used to time delays longer than 1 hour. The delay module can further include heating and cooling regions.
The substrate can further include a mixing module in fluid communication with the main channel downstream of the coalescence module and upstream of the detection module.
The substrate can further include a UV-release module in fluid communication with the main channel downstream of the inlet module and upstream of the coalescence module.
The substrate can further include a droplet spacing module in fluid communication with the main channel downstream of the inlet module to allow appropriate droplets to come with proximity for coalescence.
The continuous phase used in the channels of the microfluidic substrate can be a non-polar solvent such as, for example, a fluorocarbon oil. The continuous phase can further include one or more additives such as a surfactant or fluorosurfactant in order to stabilize the droplets. The fluorosurfactant can be a perfluorinated polyether, for example.
The dispersed phase of the microfluidic substrate can include a library of droplets of the same or different sizes (i.e., an emulsion stream) or a continuous aqueous stream. The library of droplets can include, for example, a biological or chemical material such as tissues, cells, particles, proteins, antibodies, amino acids, nucleotides, small molecules, and pharmaceuticals. The biological/chemical material can include a label such as a DNA tag, dye, a quantum dot or a radio frequency identification tag. The library of library of droplets can include a label such as a change in viscosity, a change in opacity, a change in volume, a change in density, a change in pH, a change in temperature, a change in dielectric constant, a change in conductivity, a change in the amount of beads present in the droplets, a change in the amount of flocculent in the droplets, a change in the amount of a selected solvent within the droplets or the change in the amount of any measurable entity within the droplets, or combinations thereof. A label can be detected by fluorescence polarization, fluorescence intensity, fluorescence lifetime, fluorescence energy transfer, pH, ionic content, temperature or combinations thereof.
The present invention also provides a microfluidic substrate including, for example, (i) at least one inlet module having at least one inlet channel adapted to carry at least one dispersed phase fluid; (ii) at least one main channel adapted to carry at least one continuous phase fluid, wherein the inlet channel is in fluid communication with the main channel at a junction, wherein the junction includes a fluidic nozzle designed for flow focusing such that the dispersed phase fluid is immiscible with the continuous phase fluid and forms a plurality of highly uniform, monodisperse droplets in the continuous phase fluid; (iii) at least one nanoreactor division module downstream from the inlet module wherein the main channel is divided into at least two division channels and the nanoreactor is split into at least two daughter nanoreactors; (iv) at least one second inlet channel adapted to carry at least one second dispersed phase fluid wherein the inlet channel is in fluid communication with at least one of the divisional channels at a junction, wherein the junction includes a fluidic nozzle designed for flow focusing such that the second dispersed phase fluid is immiscible with the continuous phase fluid and forms a plurality of highly uniform, monodisperse droplets in the continuous phase fluid; (v) at least one coalescence module downstream from and in fluid communication with the inlet module via the main channel including a coalescence apparatus, wherein at least one droplet from step (ii) and at least one droplet from step (iv) passing there through are coalesced; (vi) at least one reorder module downstream from the dividing module such that the daughter nanoreactors from the division channel are reordered in proximity but not coalesced; and (vii) at least one detection module downstream from the reorder module, the detection module including a detection apparatus for evaluating the contents or characteristics of at least one of nanoreactors or droplets in proximity.
The microfluidic substrate can also include a sorting module in proximity to and in fluid communication with the detection module, the sorting module including a sorting apparatus adapted to direct the droplet or nanoreactor into or away from a collection module in response to the contents or characterization of the droplet or nanoreactor evaluated in the detection module.
The detection module can evaluate the contents of two nanoreactors or droplets in proximity and the sorting module can direct the droplets or nanoreactors into or away from a collection module in response to the ratio of the contents or characterization of the droplets or nanoreactors evaluated in the detection module.
The present invention also provides a method of producing microfluidic substrate including, for example, (i) providing a base plate, wherein the base plate comprises a flat surface; (ii) providing a master including the pattern of the channels and electrodes of a microfluidic substrate; (iii) providing a molding cavity, wherein the molding cavity comprises an opening for molding an elastomeric substrate; (iv) assembling the base plate, master and molding cavity, such that the master is placed between the base plate and molding cavity and wherein the master pattern is located directly under and aligned to the opening for molding an elastomeric substrate; (v) providing a top plate containing one or more sliding molding pins used to form one or more fluid and/or electrical interconnects; (vi) assembling the top plate onto the molding cavity of step d, such that the sliding molding pins contact points on the pattern of channels and electrodes on the master; (vi) introducing a liquid elastomeric polymer into the opening on the molding cavity such that it contacts the master; (vii) solidifying the elastomeric polymer within the molding cavity; (viii) removing the solidified elastomeric polymer substrate from the top plate, bottom plate and molding cavity assembly; and (ix) bonding the solidified elastomeric polymer substrate to compatible polymeric or non-polymeric media. The sliding molding pins can be surrounded by an elastomeric sleeve. The present invention also provides a microfluidic device produces by the methods provided.
The master is generated by photolithography, photolithography and converted to a durable metal master, micromachining or by rapid prototyping methods such as stereolithography. The master can be a silicon or glass substrate patterned with photoresist. Preferably, the master is a silicon or glass substrate patterned with SU-8.
The elastomeric polymer can be a silicone elastomeric polymer. Preferably, the silicone elastreric polymer is polydimethylsiloxane. The elastomeric polymer can be solidified by curing. The elastomeric polymer can be treated with high intensity oxygen or air plasma to permit bonding to the compatible polymeric or non-polymeric media. The polymeric and non-polymeric media can be glass, silicon, silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, or epoxy polymers.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF DRAWINGS
Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying drawings, which are schematic and are not intended to be drawn to scale. In the drawings, each identical or nearly identical component illustrated is typically represented by a single numeral. For the purposes of clarity, not every component is labeled in every drawing, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the drawings:
FIG. 1 is an schematic illustrating the interacting modules of a microfluidic device of the present invention.
FIGS. 2A-B show dual and single oil versions of the nozzle concept using a small ferrule for the nozzle section. FIGS. 2C-D show the same nozzles made directly out of small bore tubing (the “nozzle” runs the entire length of the tubing).
FIG. 3 shows the expansion of the nozzle ferrule concept shown in FIGS. 2A and 2B.
FIG. 4 shows the expansion of the nozzle section contained in the ferrule.
FIG. 5A shows the operation of the nozzle in Aspiration Mode and 5B shows the operation of the nozzle in Injection Mode.
FIG. 6 shows a reservoir based sample emulsification where the well is initially filled with a fluid with lower density than the sample to be introduced.
FIG. 7 illustrates a sample introduction when the sample is less dense than the fluid in the sample port, which is an alternative scheme used to introduce samples that are less dense than the oil used to emulsify the sample.
FIG. 8 illustrates a nozzle that formed directly into the fitting used to connect the collection syringe to a syringe tip (e.g. capillary tubing) in order to create a monodisperse emulsion directly from a library well. Step 1 shows the aspiration of the sample can be accomplished by running the collection syringe in withdrawal mode at a flow rate (Q3) above the flow rate of the two oil syringes. Step 2 shows the appropriate volume of sample loaded into the capillary tubing, and the capillary tubing would be removed from the sample well, an air bubble, and possibly a cleaning solution would be aspirated. Step 3 shows when almost all of the sample has been emulsified, the collection syringe withdrawal rate would either be reduced below the oil flow rates, stopped, or set to infuse at some nominal rate.
FIGS. 9A-C illustrate a two phase system where the reagent is injected on top of the 2nd, immiscible phase. (FIG. 9A) During injection, prior to transition from 1st phase to 2nd phase. (FIG. 9B) 2nd phase just entering the transfer lines. (FIG. 9C) 2nd phase has completely filled the transfer line and pushed the entire volume of reagent through the system.
FIG. 10 illustrates sandwiching an ultra-small volume of fluid (i.e., sub-nanoliter) between two solutions having different densities.
FIG. 11 illustrates possible interconnect designs for use with PDMS devices.
FIG. 12 illustrates self-alignment of fluidic interconnect
FIG. 13 illustrates the interconnects needed for each tube molded into a single monolithic self-aligned part.
FIG. 14 shows a schematic of a molding tool based on this concept. The pins (orange) are captured within an elestomeric molded sleeve and a compression plate made from a rigid backer plate and foam rubber is used to apply gentle even pressure to the pins and generate the force needed to make the pins uniformly contact the master.
FIG. 15 is a schematic diagram of an improved coalescence module that shows an optional small constriction (neckdown) just before this expansion can be used to better align the droplets on their way into the coalescence point.
FIG. 16 illustrates that fluorescence polarization (FP) measures the tumbling rate of a compound in solution and is a function of it's volume (in most cases, volume is correlated with MW)
FIG. 17 shows the fluorescence polarization of three different compounds. Results of reading polarization in 18,000 drops containing 3 distinct species (FC, BTFC, and BTFC bound to SA). Ideal for reading results of drug screening assays, protein interactions, or DNA hybridization.
FIG. 18A illustrates encoding a liquid solution using both overall fluorescence polarization and overall dye intensity within droplets; FIG. 18B shows that multiple colors of fluorescence polarization and FI increases the number of possible labels. Ten intensity levels with ten fluorescence polarization levels on two colors yields 10,000 labels.
FIG. 19 illustrates FPcoding using dyes having different fluorescence lifetimes. These were made one element at a time, stored in a single syringe overnight and then loaded back on chip. The codes were made by using a ratio of two different dyes, one with a short lifetime and hence high FP and one with a long lifetime and correspondingly low FP. The mixtures have intermediate FP signals. The intensity is tuned by controlling the overall concentration of the two dyes.
FIG. 20A-20D illustrate the sorting and/or splitting of droplets in accordance with another embodiment of the invention
FIG. 21A-F shows the possible flow geometries used in an asymmetric sorting application.
FIGS. 22A-E show the possible electrode geometries used in an asymmetric sorting application. FIG. 22A shows the design using sharp tipped electrodes. FIG. 22B shows broad tipped electrodes to increase the interaction time between the droplets and the electric field (the tips could be many drop diameters long). FIG. 22C shows electrodes straddling the collection line. FIG. 22D shows electrodes on opposite sides of the main channel. FIG. 22E shows an Asymmetric Electrode Pair (the asymmetry may be present on any of the other electrode pair layouts as well).
FIG. 23 shows a schematic of a device that split droplets, performs different experiments on the two daughter droplets and then reorders so that they pass sequential through the detector
FIG. 24A, shows geometric parameters defining the obstacle matrix. FIG. 24B shows three fluid streams. FIG. 24C shows a particle with a radius that is larger than lane 1 follows a streamline passing through the particle's center (black dot).
FIG. 25 shows high-resolution separation of fluorescent microspheres with diameters of 0.80 um (green), 0.90 um (red), and 1.03 um (yellow), with a matrix of varying gap size.
FIG. 26 is a schematic illustrating the separation by deterministic lateral displacement in an array of microposts, with an example row shift fraction of one-third.
FIG. 27 shows a dideoxynucleotide sequencing on a microfabricated chip. Shown is one embodiment for a DNA sequencing chip design. Template DNA and primers are combined at step ‘add 1’ and the reaction is incubated at 95° C. for a hot start (position 1). The reaction then cycles 20-30 times (position 2) before the addition of SAP and Exol at ‘add 2.’ The reaction is incubated at 37° C. for a pre-defined time-period and then the SAP and Exol enzymes are inactivated at 95° C. (position ‘4’). The SAP/Exol procedure degrades nucleotides and single-stranded DNA (primers) remaining after PCR. The universal sequencing primers, ddNTPs and buffers are added at ‘add 3,’ and the PCR sequencing reaction is allowed to proceed at position ‘5.’ The final reaction product is collected and can be stored off-chip.
FIG. 28A, shows a schematic of the TempliPhi amplification process using rolling circle amplification. FIG. 28B illustrates a transcription mediated reaction. FIG. 28C illustrates strand-displacement amplification. FIG. 28D shows a schematic diagram of helicase-dependent amplification.
FIG. 29 illustrates emulsion-based sample preparation, sample preparation and DNA sequencing. Random libraries of DNA fragments are generated by shearing an entire genome and isolating single DNA molecules by limiting dilution.
FIG. 30 shows one method for isolating antibodies on a microfluidic device.
FIG. 31 shows an alternate method for isolating antibodies on a microfluidic device.
FIG. 32 shows the method of the present invention for isolating antibodies on the microfluidic device. The right panel is a diagram of individual steps proposed to amplify signal of interacting antibody and antigen. The left panel is a schematic as would be designed for a chip to be used on microfluidic device.
FIG. 33 shows the genetic selection for full length antibody clones. A genetic selection can be used to enrich for full-length antibody clones by transforming E. coli and selecting for clones able to grow on medium in which a suitable sugar is the only carbon source.
FIG. 34 is a schematic representation of a multi-step chip according to the invention. 1st stage: droplets are sent into C2, the collection port; 2nd stage: 1st stage emulsion collected into C2 is reinjected back into the chip and merged with the droplets formed in the second stage nozzle.
FIG. 35 shows karyotyping using spectral probes that allow all 23 pairs of human chromosomes to be seen at one time, with each pair of chromosomes painted in a difference fluorescent color.
FIG. 36 shows the future BioBased Economy with six building blocks based on renewable 30 biomass.
FIG. 37 shows the 8 non-renewable building blocks based on petroleum.
 DETAILED DESCRIPTION
The microfluidic devices and methods of use described herein are based on the creation and electrical manipulation of aqueous phase droplets completely encapsulated by an inert immiscible oil stream. This combination enables precise droplet generation, highly efficient, electrically addressable, droplet coalescence, and controllable, electrically addressable single droplet sorting. The microfluidic devices include one or more channels and modules. A schematic illustrating one example of interacting modules of a microfluidic substrate is shown in FIG. 1 . The integration of these modules is an essential enabling technology for a droplet based, high-throughput microfluidic reactor system.
The microfluidic devices of the present invention can be utilized for numerous biological, chemical, or diagnostic applications, as described in further detail herein.
Substrates
The microfluidic device of the present invention includes one or more analysis units. An “analysis unit” is a microsubstrate, e.g., a microchip. The terms microsubstrate, substrate, microchip, and chip are used interchangeably herein. The analysis unit includes at least one inlet channel, at least one main channel, at least one inlet module, at least one coalescence module, and at least one detection module. The analysis unit can further includes one or more sorting modules. The sorting module can be in fluid communication with branch channels which are in fluid communication with one or more outlet modules (collection module or waste module). For sorting applications, at least one detection module cooperates with at least one sorting module to divert flow via a detector-originated signal. It shall be appreciated that the “modules” and “channels” are in fluid communication with each other ‘and therefore may overlap; i.e., there may be no clear boundary where a module or channel begins or ends. A plurality of analysis units of the invention may be combined in one device. The analysis unit and specific modules are described in further detail herein.
The dimensions of the substrate are those of typical microchips, ranging between about 0.5 cm to about 15 cm per side and about 1 micron to about 1 cm in thickness. A substrate can be transparent and can be covered with a material having transparent properties, such as a glass coverslip, to permit detection of a reporter, for example, by an optical device such as an optical 25 microscope. The material can be perforated for functional interconnects, such as fluidic, electrical, and/or optical interconnects, and sealed to the back interface of the device so that the junction of the interconnects to the device is leak-proof. Such a device can allow for application of high pressure to fluid channels without leaking.
A variety of materials and methods, according to certain aspects of the invention, can be used to form any of the described components of the systems and devices of the invention. In some cases, the various materials selected lend themselves to various methods. For example, various components of the invention can be formed from solid materials, in which the channels can be formed via molding, micromachining, film deposition processes such as spin coating and chemical vapor deposition, laser fabrication, photolithographic techniques, etching methods including wet chemical or plasma processes, and the like. See, for example, Scientific American, 248:44-55, 1983 (Angell, et al). At least a portion of the fluidic system can be formed of silicone by molding a silicone chip. Technologies for precise and efficient formation of various fluidic systems and devices of the invention from silicone are known. Various components of the systems and devices of the invention can also be formed of a polymer, for example, an elastomeric polymer such as polydimethylsiloxane (“PDMS”), polytetrafluoroethylene (“PTFE”) or Teflon®, or the like.
The channels of the invention can be formed, for example by etching a silicon chip using conventional photolithography techniques, or using a micromachining technology called “soft lithography” as described by Whitesides and Xia, Angewandte Chemie International Edition 37, 550 (1998). These and other methods may be used to provide inexpensive miniaturized devices, and in the case of soft lithography, can provide robust devices having beneficial properties such as improved flexibility, stability, and mechanical strength. When optical detection is employed, the invention also provides minimal light scatter from molecule, cell, small molecule or particle suspension and chamber material.
Different components can be formed of different materials. For example, a base portion including a bottom wall and side walls can be formed from an opaque material such as silicone or PDMS, and a top portion can be formed from a transparent or at least partially transparent material, such as glass or a transparent polymer, for observation and/or control of the fluidic process. Components can be coated so as to expose a desired chemical functionality to fluids that contact interior channel walls, where the base supporting material does not have a precise, desired functionality. For example, components can be formed as illustrated, with interior channel walls coated with another material. Material used to form various components of the systems and devices of the invention, e.g., materials used to coat interior walls of fluid channels, may desirably be selected from among those materials that will not adversely affect or be affected by fluid flowing through the fluidic system, e.g., material(s) that is chemically inert in the presence of fluids to be used within the device.
Various components of the invention when formed from polymeric and/or flexible and/or elastomeric materials, and can be conveniently formed of a hardenable fluid, facilitating formation via molding (e.g. replica molding, injection molding, cast molding, etc.). The hardenable fluid can be essentially any fluid that can be induced to solidify, or that spontaneously solidifies, into a solid capable of containing and/or transporting fluids contemplated for use in and with the fluidic network. In one embodiment, the hardenable fluid comprises a polymeric liquid or a liquid polymeric precursor (i.e. a “prepolymer”). Suitable polymeric liquids can include, for example, thermoplastic polymers, thermoset polymers, or mixture of such polymers heated above their melting point. As another example, a suitable polymeric liquid may include a solution of one or more polymers in a suitable solvent, which solution forms a solid polymeric material upon removal of the solvent, for example, by evaporation. Such polymeric materials, which can be solidified from, for example, a melt state or by solvent evaporation, are well known to those of ordinary skill in the art. A variety of polymeric materials, many of which are elastomeric, are suitable, and are also suitable for forming molds or mold masters, for embodiments where one or both of the mold masters is composed of an elastomeric material. A non-limiting list of examples of such polymers includes polymers of the general classes of silicone polymers, epoxy polymers, and acrylate polymers. Epoxy polymers are characterized by the presence of a three-membered cyclic ether group commonly referred to as an epoxy group, 1,2-epoxide, or oxirane. For example, diglycidyl ethers of bisphenol A can be used, in addition to compounds based on aromatic amine, triazine, and cycloaliphatic backbones. Another example includes the well-known Novolac polymers. Non-limiting examples of silicone elastomers suitable for use according to the invention include those formed from precursors including the chlorosilanes such as methylchlorosilanes, ethylchlorosilanes, phenylchlorosilanes, etc.
Silicone polymers are preferred, for example, the silicone elastomer polydimethylsiloxane. Non-limiting examples of PDMS polymers include those sold under the trademark Sylgard by Dow Chemical Co., Midland, Mich., and particularly Sylgard 182, Sylgard 184, and Sylgard 186. Silicone polymers including PDMS have several beneficial properties simplifying formation of the microfluidic structures of the invention. For instance, such materials are inexpensive, readily available, and can be solidified from a prepolymeric liquid via curing with heat. For example, PDMSs are typically curable by exposure of the prepolymeric liquid to temperatures of about, for example, about 65° C. to about 75° C. for exposure times of, for example, about an hour. Also, silicone polymers, such as PDMS, can be elastomeric and thus may be useful for forming very small features with relatively high aspect ratios, necessary in certain embodiments of the invention. Flexible (e.g., elastomeric) molds or masters can be advantageous in this regard.
The present invention provides improved methods of bonding PDMS to incompatible media. Normal methods of bonding various materials (plastic, metals, etc) directly to materials such as PDMS, silicone, Teflon, and PEEK using traditional bonding practices (adhesives, epoxies, etc) do not Work well due to the poor adhesion of the bonding agent to materials such as PDMS. Normal surface preparation by commercially available surface activators has not worked well in microfluidic device manufacturing. This problem is eliminated by treating the PDMS surface to be bonded with high intensity oxygen or air plasma. The process converts the top layer of PDMS to glass which bonds extremely well with normal adhesives. Tests using this method to bond external fluid lines to PDMS using a UV-cure adhesive (Loctite 352, 363, and others) resulted in a bond that is stronger than the PDMS substrate, resulting in fracture of the PDMS prior to failure of the bond. The present method combines high radiant flux, wavelength selection, and cure exposure time to significantly enhance the bond strength of the adhesive.
One advantage of forming structures such as microfluidic structures of the invention from silicone polymers, such as PDMS, is the ability of such polymers to be oxidized, for example by exposure to an oxygen-containing plasma such as an air plasma, so that the oxidized structures contain, at their surface, chemical groups capable of cross-linking to other oxidized silicone polymer surfaces or to the oxidized surfaces of a variety of other polymeric and non-polymeric materials. Thus components can be formed and then oxidized and essentially irreversibly sealed to other silicone polymer surfaces, or to the surfaces of other substrates reactive with the oxidized silicone polymer surfaces, without the need for separate adhesives or other sealing means. In most cases, sealing can be completed simply by contacting an oxidized silicone surface to another surface without the need to apply auxiliary pressure to form the seal. That is, the pre-oxidized silicone surface acts as a contact adhesive against suitable mating surfaces. Specifically, in addition to being irreversibly sealable to itself, oxidized silicone such as oxidized PDMS can also be sealed irreversibly to a range of oxidized materials other than itself including, for example, glass, silicon, silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, and epoxy polymers, which have been oxidized in a similar fashion to the PDMS surface (for example, via exposure to an oxygen-containing plasma). methods useful in the context of the present invention, as well as overall molding techniques, are described in the art, for example, in an article entitled “Rapid Prototyping of Microfluidic Systems and Polydimethylsiloxane,” Anal. Chem., 70:474-480, 1998 (Duffy et al.), incorporated herein by reference.
Another advantage to forming microfluidic structures of the invention (or interior, fluid contacting surfaces) from oxidized silicone polymers is that these surfaces can be much more hydrophilic than the surfaces of typical elastomeric polymers (where a hydrophilic interior surface is desired). Such hydrophilic channel surfaces can thus be more easily filled and wetted with aqueous solutions than can structures comprised of typical, unoxidized elastomeric polymers or other hydrophobic materials.
In one embodiment, a bottom wall is formed of a material different from one or more side walls or a top wall, or other components. For example, the interior surface of a bottom wall can comprise the surface of a silicon wafer or microchip, or other substrate. Other components can, as described above, be sealed to such alternative substrates. Where it is desired to seal a component comprising a silicone polymer (e.g. PDMS) to a substrate (bottom wall) of different material, the substrate may be selected from the group of materials to which oxidized silicone polymer is able to irreversibly seal (e.g., glass, silicon, silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, epoxy polymers, and glassy carbon surfaces which have been oxidized). Alternatively, other sealing techniques can be used, as would be apparent to those of ordinary skill in the art, including, but not limited to, the use of separate adhesives, thermal bonding, solvent bonding, ultrasonic welding, etc.
Channels
The microfluidic substrates of the present invention include channels that form the boundary for a fluid. A “channel,” as used herein, means a feature on or in a substrate that at least partially directs the flow of a fluid. In some cases, the channel may be formed, at least in part, by a single component, e.g., an etched substrate or molded unit. The channel can have any cross-sectional shape, for example, circular, oval, triangular, irregular, square or rectangular (having any aspect ratio), or the like, and can be covered or uncovered (i.e., open to the external environment surrounding the channel). In embodiments where the channel is completely covered, at least one portion of the channel can have a cross-section that is completely enclosed, and/or the entire channel may be completely enclosed along its entire length with the exception of its inlet and outlet.
An open channel generally will include characteristics that facilitate control over fluid transport, e.g., structural characteristics (an elongated indentation) and/or physical or chemical characteristics (hydrophobicity vs. hydrophilicity) and/or other characteristics that can exert a force (e.g., a containing force) on a fluid. The fluid within the channel may partially or completely fill the channel. In some cases the fluid may be held or confined within the channel or a portion of the channel in some fashion, for example, using surface tension (e.g., such that the fluid is held within the channel within a meniscus, such as a concave or convex meniscus). In an article or substrate, some (or all) of the channels may be of a particular size or less, for example, having a largest dimension perpendicular to fluid flow of less than about 5 mm, less than about 2 mm, less than about 1 mm, less than about 500 microns, less than about 200 microns, less than about 100 microns, less than about 60, less than about 50 microns, less than about 40 microns, less than about 30 microns, less than about 25 microns, less than about 10 microns, less than about 3 microns, less than about 1 micron, less than about 300 nm, less than about 100 nm, less than about 30 nm, or less than about 10 nm or less in some cases. Of course, in some cases, larger channels, tubes, etc. can be used to store fluids in bulk and/or deliver a fluid to the channel. In one embodiment, the channel is a capillary.
The dimensions of the channel may be chosen such that fluid is able to freely flow through the channel, for example, if the fluid contains cells. The dimensions of the channel may also be chosen, for example, to allow a certain volumetric or linear flow rate of fluid in the channel. Of course, the number of channels and the shape of the channels can be varied by any method known to those of ordinary skill in the art. In some cases, more than one channel or capillary may be used. For example, two or more channels may be used, where they are positioned inside each other, positioned adjacent to each other, etc.
For particles (e.g., cells) or molecules that are in droplets (i.e., deposited by the inlet module) within the flow of the main channel, the channels of the device are preferably square, with a diameter between about 2 microns and 1 mm. This geometry facilitates an orderly flow of droplets in the channels. Similarly, the volume of the detection module in an analysis device is typically in the range of between about 0.1 picoliters and 500 nanoliters.
A “main channel” is a channel of the device of the invention which permits the flow of molecules, cells, small molecules or particles past a coalescence module for coalescing one or more droplets, a detection module for detection (identification) or measurement of a droplet and a sorting module, if present, for sorting a droplet based on the detection in the detection module. The main channel is typically in fluid communication with the coalescence, detection and/or sorting modules, as well as, an inlet channel of the inlet module. The main channel is also typically in fluid communication with an outlet module and optionally with branch channels, each of which may have a collection module or waste module. These channels permit the flow of molecules, cells, small molecules or particles out of the main channel. An “inlet channel” permits the flow of molecules, cells, small molecules or particles into the main channel. One or more inlet channels communicate with one or more means for introducing a sample into the device of the present invention. The inlet channel communicates with the main channel at an inlet module.
The microfluidic substrate can also comprise one or more fluid channels to inject or remove fluid in between droplets in a droplet stream for the purpose of changing the spacing between droplets.
The channels of the device of the present invention can be of any geometry as described. However, the channels of the device can comprise a specific geometry such that the contents of the channel are manipulated, e.g., sorted, mixed, prevent clogging, etc.
A microfluidic substrate can also include a specific geometry designed in such a manner as to prevent the aggregation of biological/chemical material and keep the biological/chemical material separated from each other prior to encapsulation in droplets. The geometry of channel dimension can be changed to disturb the aggregates and break them apart by various methods, that can include, but is not limited to, geometric pinching (to force cells through a (or a series of) narrow region(s), whose dimension is smaller or comparable to the dimension of a single cell) or a barricade (place a series of barricades on the way of the moving cells to disturb the movement and break up the aggregates of cells).
To prevent material (e.g., cells and other particles or molecules) from adhering to the sides of the channels, the channels (and coverslip, if used) may have a coating which minimizes adhesion. Such a coating may be intrinsic to the material from which the device is manufactured, or it may be applied after the structural aspects of the channels have been microfabricated. “TEFLON” is an example of a coating that has suitable surface properties. The surface of the channels of the microfluidic device can be coated with any anti-wetting or blocking agent for the dispersed phase. The channel can be coated with any protein to prevent adhesion of the biological/chemical sample. For example, in one embodiment the channels are coated with BSA, PEG-silane and/or fluorosilane. For example, 5 mg/ml BSA is sufficient to prevent attachment and prevent clogging. In another embodiment, the channels can be coated with a cyclized transparent optical polymer obtained by copolymerization of perfluoro (alkenyl vinyl ethers), such as the type sold by Asahi Glass Co. under the trademark Cytop. In such an embodiment, the coating is applied from a 0.1-0.5 wt % solution of Cytop CTL-809M in CT-Solv 180. This solution can be injected into the channels of a microfluidic device via a plastic syringe. The device can then be heated to about 90° C. for 2 hours, followed by heating at 200° C. for an additional 2 hours. In another embodiment, the channels can be coated with a hydrophobic coating of the type sold by PPG Industries, Inc. under the trademark Aquapel (e.g., perfluoroalkylsilane surface treatment of plastic and coated plastic substrate surfaces in conjunction with the use of a silica primer layer) and disclosed in U.S. Pat. No. 5,523,162, which patient is hereby incorporated by reference. By fluorinating the surfaces of the channels, the continuous phase preferentially wets the channels and allows for the stable generation and movement of droplets through the device. The low surface tension of the channel walls thereby minimizes the accumulation of channel clogging particulates.
The surface of the channels in the microfluidic device can be also fluorinated to prevent undesired wetting behaviors. For example, a microfluidic device can be placed in a polycarbonate dessicator with an open bottle of (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane. The dessicator is evacuated for 5 minutes, and then sealed for 20-40 minutes. The dessicator is then backfilled with air and removed. This approach uses a simple diffusion mechanism to enable facile infiltration of channels of the microfluidic device with the fluorosilane and can be readily scaled up for simultaneous device fluorination.
Fluids
The microfluidic device Of the present invention is capable of controlling the direction and flow of fluids and entities within the device. The term “flow” means any movement of liquid or solid through a device or in a method of the invention, and encompasses without limitation any fluid stream, and any material moving with, within or against the stream, whether or not the material is carried by the stream. For example, the movement of molecules, beads, cells or virions through a device or in a method of the invention, e.g. through channels of a microfluidic chip of the invention, comprises a flow. This is so, according to the invention, whether or not the molecules, beads, cells or virions are carried by a stream of fluid also comprising a flow, or whether the molecules, cells or virions are caused to move by some other direct or indirect force or motivation, and whether or not the nature of any motivating force is known or understood. The application of any force may be used to provide a flow, including without limitation, pressure, capillary action, electro-osmosis, electrophoresis, dielectrophoresis, optical tweezers, and combinations thereof, without regard for any particular theory or mechanism of action, so long as molecules, cells or virions are directed for detection, measurement or sorting according to the invention. Specific flow forces are described in further detail herein.
The flow stream in the main channel is typically, but not necessarily, continuous and may be stopped and started, reversed or changed in speed. A liquid that does not contain sample molecules, cells or particles can be introduced into a sample inlet well or channel and directed through the inlet module, e.g., by capillary action, to hydrate and prepare the device for use. Likewise, buffer or oil can also be introduced into a main inlet region that communicates directly with the main channel to purge the device (e.g., or “dead” air) and prepare it for use. If desired, the pressure can be adjusted or equalized, for example, by adding buffer or oil to an outlet module.
As used herein, the term “fluid stream” or “fluidic stream” refers to the flow of a fluid, typically generally in a specific direction. The fluidic stream may be continuous and/or discontinuous. A “continuous” fluidic stream is a fluidic stream that is produced as a single entity, e.g., if a continuous fluidic stream is produced from a channel, the fluidic stream, after production, appears to be contiguous with the channel outlet. The continuous fluidic stream is also referred to as a continuous phase fluid or carrier fluid. The continuous fluidic stream may be laminar, or turbulent in some cases.
Similarly, a “discontinuous” fluidic stream is a fluidic stream that is not produced as a single entity. The discontinuous fluidic stream is also referred to as the dispersed phase fluid or sample fluid. A discontinuous fluidic stream may have the appearance of individual droplets, optionally surrounded by a second fluid. A “droplet,” as used herein, is an isolated portion of a first fluid that completely surrounded by a second fluid. In some cases, the droplets may be spherical or substantially spherical; however, in other cases, the droplets may be spherical or substantially spherical; however, in other cases, the droplets may be non-spherical, for example, the droplets may have the appearance of “blobs” or other irregular shapes, for instance, depending on the external environment. As used herein, a first entity is “surrounded” by a second entity if a closed loop can be drawn or idealized around the first entity through only the second entity. The dispersed phase fluid can include a biological/chemical material. The biological/chemical material can be tissues, cells, particles, proteins, antibodies, amino acids, nucleotides, small molecules, and pharmaceuticals. The biological/chemical material can include one or more labels known in the art. The label can be a DNA tag, dyes or quantum dot, or combinations thereof.
Droplets
The term “emulsion” refers to a preparation of one liquid distributed in small globules (also referred to herein as drops, droplets or NanoReactors) in the body of a second liquid. The first and second fluids are immiscible with each other. For example, the discontinuous phase can be an aqueous solution and the continuous phase can a hydrophobic fluid such as an oil. This is termed a water in oil emulsion. Alternatively, the emulsion may be a oil in water emulsion. In that example, the first liquid, which is dispersed in globules, is referred to as the discontinuous phase, whereas the second liquid, is referred to as the continuous phase or the dispersion medium. The continuous phase can be an aqueous solution and the discontinuous phase is a hydrophobic fluid, such as an oil (e.g., decane, tetradecane, or hexadecane). The droplets or globules of oil in an oil in water emulsion are also referred to herein as “micelles”, whereas globules of water in a water in oil emulsion may be referred to as “reverse micelles”.
The fluidic droplets may each be substantially the same shape and/or size. The shape and/or size can be determined, for example, by measuring the average diameter or other characteristic dimension of the droplets. The “average diameter” of a plurality or series of droplets is the arithmetic average of the average diameters of each of the droplets. Those of ordinary skill in the art will be able to determine the average diameter (or other characteristic dimension) of a plurality or series of droplets, for example, using laser light scattering, microscopic examination, or other known techniques. The diameter of a droplet, in a non-spherical droplet, is the mathematically-defined average diameter of the droplet, integrated across the entire surface. The average diameter of a droplet (and/or of a plurality or series of droplets) may be, for example, less than about 1 mm, less than about 500 micrometers, less than about 200 micrometers, less than about 100 micrometers, less than about 75 micrometers, less than about 50 micrometers, less than about 25 micrometers, less than about 10 micrometers, or less than about 5 micrometers in some cases. The average diameter may also be at least about 1 micrometer, at least about 2 micrometers, at least about 3 micrometers, at least about 5 micrometers, at least about 10 micrometers, at least about 15 micrometers, or at least about 20 micrometers in certain cases.
As used herein, the term “NanoReactor” and its plural encompass the terms “droplet”, “nanodrop”, “nanodroplet”, “microdrop” or “microdroplet” as defined herein, as well as an integrated system for the manipulation and probing of droplets, as described in detail herein. Nanoreactors as described herein can be 0.1-1000 μm (e.g., 0.1, 0.2 . . . 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 . . . 1000), or any size within in this range. Droplets at these dimensions tend to conform to the size and shape of the channels, while maintaining their respective volumes. Thus, as droplets move from a wider channel to a narrower channel they become longer and thinner, and vice versa.
The microfluidic substrate of this invention most preferably generate round, monodisperse droplets. The droplets can have a diameter that is smaller than the diameter of the microchannel; i.e., preferably 15 to 100 μm when cells are used; or 10 to 75 μm when reagents or other chemical or biological agents are used; or 100 to 1000 μm when droplets are used for sequencing reactions such that droplets will be removed and dispensed into other collection apparatuses, such as microtiter plates or utilized in sequencing devices. Monodisperse droplets are particularly preferably, e.g., in high throughput devices and other embodiments where it is desirable to generate droplets at high frequency and of high uniformity.
The droplet forming liquid is typically an aqueous buffer solution, such as ultrapure water (e.g., 18 mega-ohm resistivity, obtained, for example by column chromatography), 10 mM Tris HCl and 1 mM EDTA (TE) buffer, phosphate buffer saline (PBS) or acetate buffer. Any liquid or buffer that is physiologically compatible with the population of molecules, cells or particles to be analyzed and/or sorted can be used. The fluid passing through the main channel and in which the droplets are formed is one that is immiscible with the droplet forming fluid. The fluid passing through the main channel can be a non-polar solvent, decane (e g., tetradecane or hexadecane), fluorocarbon oil, silicone oil or another oil (for example, mineral oil).
The dispersed phase fluid may also contain biological/chemical material (e.g., molecules, cells or other particles) for combination, analysis and/or sorting in the device. The droplets of the dispersed phase fluid can contain more than one particle or can contain no more than one particle. For example, where the biological material comprises cells, each droplet preferably contains, on average, no more than one cell. However, in some embodiments, each droplet may contain, on average, at least 1000 cells. The droplets can be detected and/or sorted according to their contents.
The concentration (i.e., number) of molecules, cells or particles in a droplet can influence sorting efficiently and therefore is preferably optimized. In particular, the sample concentration should be dilute enough that most of the droplets contain no more than a single molecule, cell or particle, with only a small statistical chance that a droplet will contain two or more molecules, cells or particles. This is to ensure that for the large majority of measurements, the level of reporter measured in each droplet as it passes through the detection module corresponds to a single molecule, cell or particle and not to two or more molecules, cells or particles.
The parameters which govern this relationship are the volume of the droplets and the concentration of molecules, cells or particles in the sample solution. The probability that a droplet will contain two or more molecules, cells or particles (P≤2) can be expressed as
P ≤2=1−{1+[cell]×V}×e −[cell]×V
where “[cell]” is the concentration of molecules, cells or particles in units of number of molecules, cells or particles per cubic micron (μm3), and V is the volume of the droplet in units of μm3.
It will be appreciated that P≤2 can be minimized by decreasing the concentration of molecules, cells or particles in the sample solution. However, decreasing the concentration of molecules, cells or particles in the sample solution also results in an increased volume of solution processed through the device and can result in longer run times. Accordingly, it is desirable to minimize to presence of multiple molecules, cells or particles in the droplets (thereby increasing the accuracy of the sorting) and to reduce the volume of sample, thereby permitting a sorted sample in a reasonable time in a reasonable volume containing an acceptable concentration of molecules, cells or particles.
The maximum tolerable P≤2 depends on the desired “purity” of the sorted sample. The “purity” in this case refers to the fraction of sorted molecules, cells or particles that posses a desired characteristic (e.g., display a particular antigen, are in a specified size range or are a particular type of molecule, cell or particle). The purity of the sorted sample is inversely proportional to P≤2. For example, in applications where high purity is not needed or desired a relatively high P≤2 (e.g., P≤2=0.2) may be acceptable. For most applications, maintaining P≤2 at or below about 0.1, preferably at or below about 0.01, provides satisfactory results.
The fluids used to generate droplets in microfluidic devices are typically immiscible liquids such as oil and water. These two materials generally have very different dielectric constants associated with them. These differences can be exploited to determine droplet rate and size for every drop passing through a small section of a microfluidic device. One method to directly monitor this variation in the dielectric constant measures the change in capacitance over time between a pair of closely spaced electrodes. This change in capacitance can be detected by the change in current measured in these electrodes:
i = V ⁢ dC dt
Where i is the current, V is the voltage applied across the electrodes, and dC/dt is the change in capacitance with time. Alternatively, the capacitance can be measured directly if a time varying voltage is applied to these same electrodes: i=CdV/dt Where C is the measured capacitance, and dV/dt is the change in voltage with time. As a first approximation, the electrode pair can be determined as a parallel plate capacitor:
C = ɛ 0 ⁢ k ⁢ A d
Where ε0 is the permittivity of free space, k is the effective dielectric constant (this changes every time a droplet passes through), A is the area of the capacitor and d is the electrode separation. The current measured in the device is then plotted as a function of time.
The fluidic droplets may contain additional entities, for example, other chemical, biochemical, or biological entities (e.g., dissolved or suspended in the fluid), cells, particles, gases, molecules, or the like. In some cases, the droplets may each be substantially the same shape or size, as discussed above. In certain instances, the invention provides for the production of droplets consisting essentially of a substantially uniform number of entities of a species therein (i.e., molecules, cells, particles, etc.). For example, about 90%, about 93%, about 95%, about 97%, about 98%, or about 99%, or more of a plurality or series of droplets may each contain the same number of entities of a particular species. For instance, a substantial number of fluidic droplets produced, e.g., as described above, may each contain 1 entity, 2 entities, 3 entities, 4 entities, 5 entities, 7 entities, 10 entities, 15 entities, 20 entities, 25 entities, 30 entities, entities, 50 entities, 60 entities, 70 entities, 80 entities, 90 entities, 100 entities, etc., where the entities are molecules or macromolecules, cells, particles, etc. In some cases, the droplets may each independently contain a range of entities, for example, less than 20 entities, less than 15 entities, less than 10 entities, less than 7 entities, less than 5 entities, or less than 3 entities in some cases. In some embodiments, a droplet may contain 100,000,000 entities. In other embodiments, a droplet may contain 1,000,000 entities.
In a liquid containing droplets of fluid, some of which contain a species of interest and some of which do not contain the species of interest, the droplets of fluid may be screened or sorted for those droplets of fluid containing the species as further described below (e.g., using fluorescence or other techniques such as those described above), and in some cases, the droplets may be screened or sorted for those droplets of fluid containing a particular number or range of entities of the species of interest, e.g., as previously described. Thus, in some cases, a plurality or series of fluidic droplets, some of which contain the species and some of which do not, may be enriched (or depleted) in the ratio of droplets that do contain the species, for example, by a factor of at least about 2, at least about 3, at least about 5, at least about 10, at least about 15, at least about 20, at least about 50, at least about 100, at least about 125, at least about 150, at least about 200, at least about 250, at least about 500, at least about 750, at least about 1000, at least about 2000, or at least about 5000 or more in some cases. In other cases, the enrichment (or depletion) may be in a ratio of at least about 104, at least about 105, at least about 106, at least about 107, at least about 108, at least about 109, at least about 1010, at least about 1011, at least about 1012; at least about 1013, at least about 1014, at least about 1015, or more. For example, a fluidic droplet containing a particular species may be selected from a library of fluidic droplets containing various species, where the library may have about 100, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 1010, about 1011, about 1012, about 1013, about 1014, about 1015, or more items, for example, a DNA library, an RNA library, a protein library, a combinatorial chemistry library, etc. In certain embodiments, the droplets carrying the species may then be fused, reacted, or otherwise used or processed, etc., as further described below, for example, to initiate or determine a reaction.
Droplets of a sample fluid can be formed within the inlet module on the microfluidic device or droplets (or droplet libraries) can be formed before the sample fluid is introduced to the microfluidic device (“off chip” droplet formation). To permit effective interdigitation, coalescence and detection, the droplets comprising each sample to be analyzed must be monodisperse. As described in more detail herein, in many applications, different samples to be analyzed are contained within droplets of different sizes. Droplet size must be highly controlled to ensure that droplets containing the correct contents for analysis and coalesced properly. As such, the present invention provides devices and methods for forming droplets and droplet libraries.
Devices and Methods for Forming Sample Droplets on a Microfluidic Substrate
The present invention provides compositions and methods for forming sample droplet emulsions on a microfluidic substrate. The present invention also provides embedded microfluidic nozzles. In order to create a monodisperse emulsion directly from a library well, this invention would form a nozzle directly into the fitting used to connect the storage well/reservoir (e.g. syringe) to a syringe tip (e.g. capillary tubing), as shown in FIGS. 2-6 . FIG. 2 , Panels A and B, show dual and single oil versions of the nozzle concept using a small ferrule for the nozzle section. FIG. 2 , Panels C and D, show the same Nozzles made directly out of small bore tubing (the “nozzle” runs the entire length of the tubing). Both designs can form droplets identically, although the pressure drop will be higher for the tube based nozzle (bottom). FIG. 3 shows the expansion of the nozzle ferrule concept shown in FIGS. 2A and 2B. The tube based nozzles (FIG. 2C, 2D) function identically to this, except the “nozzle” runs the entire length of the tube instead of having a short transition. The ability to form droplets is identical in both cases. FIG. 4 shows the expansion of the nozzle section contained in the ferrule. The tee design in FIG. 2D has been built and tested, with a cross-section cut of this design shown in FIG. 5 . FIG. 5A shows the operation of the nozzle in Aspiration Mode and FIG. 5B shows the operation of the nozzle in Injection Mode. The droplets formed are approximately 45 um in diameter, and were formed from PCR mix (210 ul/hr) and SpectraSyn-10 (600 ul/hr). Other tests have been demonstrated with Spectrasyn-2 and PCR. mix. The droplets are traveling in 300 um wide×260 um deep channels. The Nozzle tube used was 100 um in diameter, and the fluids used were PCR Mix and Spectrasyn-10 with surfactant.
Since the flow is three dimensional, under this design surface wetting effects are minimized. The nozzle can be made from one or two oil lines providing constant flow of oil into the nozzle, a connection to the capillary tubing, and a connection to the storage well/reservoir (e.g. syringe). The high resolution part of the nozzle can be made out of a small bore tubing or a small, simple part molded or stamped from an appropriate material (Teflon®, plastic, metal, etc). If necessary, the nozzle itself could be formed into the tip of the ferrule using post mold processing such as laser ablation or drilling.
This nozzle design eliminates the surface wetting issues surrounding the quasi-2D flow associated with typical microfluidic nozzles made using soft lithography or other standard microfluidic chip manufacturing techniques. This is because the nozzle design is fully 3-dimensional, resulting is a complete isolation of the nozzle section from the continuous aqueous phase. This same design can also be used for generation of emulsions required for immediate use, where the aqueous line would be attached directly to a syringe and the outlet of the nozzle would be used to transport the emulsion to the point’ of use (e.g. into a microfluidic PCR chip, delay line, etc).
In another embodiment, the present invention provides compositions and methods to directly emulsify library elements from standard library storage geometries (e.g. 96 well plates, etc). In order to create a monodisperse emulsion from fluids contained in a library well plate, this invention would include microfluidic based nozzles manufactured simultaneously with an appropriately designed fluidic interconnect or well. FIGS. 6 and 7 present two possible approaches to interface with the nozzle.
FIG. 6 shows a reservoir based sample emulsification. In FIG. 6 , the well is initially filled with a fluid with lower density than the sample to be introduced. The operation of this device would be very similar to the device described above, with the exception that the sample would be introduced into a port instead of being directly aspirated from a sample well. This could either be emulsification oil obtained directly from the nozzle, or a different material that is loaded or flowing into the well automatically. The oil lines would begin flowing at their prescribed rates (FIG. 6 e ), while the collection or waste port would begin withdrawing at a rate corresponding to the total oil flow plus the desired sample flow. Once the flow has been established, the sample would be introduced into the port either manually (e.g. a pipette) or with a robotic sample handling system. The sample volume permitted would be dependent on the port volume. Since the sample is more dense than the fluid in the well, it would settle into the bottom of the well and be transported to the nozzle (FIGS. 6 a-6 d ). During this time, either the waste (used during startup only if transients cause problems) or the collection port would be withdrawing emulsified sample and stored. When the sample is completely emulsified the next sample would be introduced and the process repeated. If washing steps are required between runs, the washing fluids would be withdrawn into the waste line. If the pressure drop across the nozzle would cause cavitation on collection then an optional pressurization of the input well can be utilized.
FIG. 7 shows sample introduction when the sample is less dense than the fluid in the sample port. FIG. 7 depicts an alternative scheme that could be used to introduce samples that are less dense than the oil used to emulsify the sample. As with the concept in FIG. 1 , the process of introducing the sample into the port could be run either manually or with a robotic sampling system. In this concept, the sample port could be filled with the emulsification oil through backflow from the nozzle prior to introduction of the sample (FIG. 7 a ). If this oil is not appropriate, the port can be filled from the top with a different immiscible fluid that might have more desirable properties than the emulsification oil (e.g. better wetting, less surfactant, etc). This second immiscible fluid could be introduced during startup and flow continuously into the port when the sample tip is not inserted. Keeping the sample port filled with fluid will prevent air entrainment during startup and should improve transient performance.
If the sample tip is connected to a pump capable of driving the sample into the device, it could be started up as the tip is inserted into the device (7 b-c). When used with this sort of sample introduction, the device could be run identically to the “normal” operation of our devices, including having the “transport to waste” line (7 e) not connected to a pump. If the sample tip loading pump is not capable of accurately forcing the flow (i.e. not connected to a suitable pump), the back end of the tip could be connected to a valve that would open to either atmospheric pressure (or possibly a pressurized gas supply) when the tip is fully inserted into the port. In order to prevent air entrainment into the sample tip and device, this connection could be made through a reservoir filled with the desired immiscible liquid. In either case, the device would run identically to the one described above and shown in FIG. 6 . FIG. 7 also shows another possible configuration of the aspiration probe assembly used for the device in FIG. 6 .
Methods for Forming Sample Droplet Emulsions Prior to Injection on a Microfluidic Substrate
The present invention also provides compositions and methods for creating emulsion of the sample fluid (e.g. droplets) prior to the introduction of the sample fluid into the microfluidic devices of the present invention. More specifically, the methods are directed to the creating sample droplet emulsions “off chip”, for example in a syringe. In order to create a monodisperse emulsion directly from a library well, a nozzle is formed directly into the fitting used to connect the collection syringe to a syringe tip (e.g. capillary tubing), as shown in FIG. 8 . The nozzle can be made from one or two oil lines providing constant flow of oil into the nozzle, a connection to the capillary tubing, and a connection to the collection syringe. Aspiration of the sample can be accomplished by running the collection syringe in withdrawal mode at a flow rate (Q3) above the flow rate of the two oil syringes (Step 1 in FIG. 8 ). The difference in flow would correspond to the flow rate aspirated from the sample well. When the appropriate volume of sample has been loaded into the capillary tubing, the capillary tubing would be removed from the sample well, an air bubble, and possibly a cleaning solution would be aspirated (Step 2 in FIG. 8 ). When almost all of the sample has been emulsified, the collection syringe withdrawal rate would either be reduced below the oil flow rates, stopped, or set to infuse at some nominal rate (Step 3 in FIG. 8 ). The remaining sample, air, cleaning solution, etc, left in the capillary would be flushed back out into a cleaning well and the outside of the capillary would be cleaned at the “wash station.” When the capillary is completely clean, the process would repeat for the next library element.
The nozzle can be formed through using small bore tubing (glass, Teflon®, PEEK tubing or capillaries) or micro-fabrication or molding processes such as PDMS soft lithography, glass etching, hot embossing, or similar high resolution fabrication technology.
The present apparatus can be readily adapted for clinical applications or work where cross contamination must be eliminated, since the region from the nozzle to the syringe are isolated from the sample stream (e.g., the oil wets these surfaces and keeps the sample from directly contacting aqueous sample). The aspiration tip can be designed as a disposable item (like a robotic sampler aspiration tips) and automatically replaced between samples.
Multiple nozzle/syringe pairs can be operated in parallel, thus increasing throughput. This allows simultaneous sampling of multiple wells/samples during a single process step. Each sample can be collected into a separate syringe.
Other methods for forming sample droplet emulsions and emulsion libraries “off chip” are described in Example 1.
Methods for Minimizing Sample Volume Loss
As described herein, a significant problem when working with very small amounts of reagents comes from the losses associated with dead volumes found in the storage containers and transport lines. As an example, if 50 microliters of a material is injected into a 254 micron internal diameter capillary tube, a 75 cm long tube would consume about 38 microliters of the material (˜75%). To address this problem, the present invention provides compositions and methods which eliminates the problems associated with dead volume and reagent waste when working with extremely small volumes of reagents.
In one embodiment, the primary reagents (sample) is combined with a second, immiscible phase in the storage container (e.g. a syringe or other reservoir). This second phase is used to push the entire amount of the first phase into the system with no significant losses. More specifically, when two immiscible fluids are combined in a reservoir, the two fluids will tend to separate into layers as long as the densities of the materials are different. If the fluid of interest (e.g., sample fluid) is closest to the exit of the reservoir, it will be the first to leave when the reservoir is emptied (the exit can be on either the top or bottom, depending on the density difference). Once the reagent has been pumped out of the reservoir, the second phase will follow. This second phase will then push the first phase completely through the system without any sample fluid loss. As an example of this, oil and water (the reagent) would be combined in a syringe. If the oil is denser than the water, the syringe would be oriented with its exit face up, if the oil were less dense, then the syringe would be face down. The oil would be chosen such that the materials of interest in the reagent are not soluble in the oil phase. FIG. 9 is one example of this approach when a syringe is used as the reservoir and the second phase is denser than the reagent phase. If the reagent were more dense, then the syringe orientation would be reversed (i.e. the exit would be facing downward in the figure). Specifically, FIG. 9 shows a two phase system where the reagent is injected on top of the second, immiscible phase: (A) During injection, prior to transition from first phase to second phase, (B) second phase just entering the transfer line, (C) second phase has completely filled the transfer line and pushed the entire volume of reagent through the system.
Alternatively, a sample solution is sandwiched between two immiscible liquids, wherein one liquid has a density greater than the sample density, and the second liquid has a density less than the sample density. Referring to FIG. 10 , the sample (density 1.0) can be layered between perflourocarbon oil (density 1.8) and mineral oil (density 0.914). Ideally, when the device is being used to analyze biological reactions the immiscible solutions do not inhibit the reactions of the sample, nor are any test molecules in the sample or the sample itself soluble in either immiscible fluid. The less dense fluid (in FIG. 10 , the mineral oil) can be used to ‘prime the pump’ and remove any air or dead-space that occurs during normal injection. The sample then rises to the injection point after the mineral oil. It is further contemplated that the methods disclosed herein would also work for gases. The gases and/or liquids can be miscible, but of different densities such that they are layered on top each other in a manner that prevents their mixing.
Solid or Semi-Solid Phase Droplets
The present invention also provides solid phase particles and methods for the forming solid phase particles on a microfluidic device for downstream analysis. The solid phase particles can be used for various biological or chemical analysis (e.g., DNA or protein analyses).
For DNA analysis, post amplification encapsulation of amplicons occurs within a gel or polymer matrix prior to breaking of the droplet emulsion. Amplification reactions within droplets using one of several amplification type methods (described in further detail herein), including, but not limited to; PCR, Rolling Circle Amplification (RCA), Nucleic Acid Sequence Based Amplification (NASBA), ligase chain reaction, etc. followed by encapsulation/solidification of the amplified reaction within the droplets by either polymerizing the droplets using chemical or physical means.
A physical means might be termed ‘gelling’ whereby one incorporates low temperature agarose within the droplet during formulation and keeping the droplet above the solidification temperature until one desires the droplet to solidify.
A chemical means might be termed ‘polymerization’ whereby one combines (if needed) the droplet with a polymerizing solution and then polymerizing the droplet using either a polymerization initiator (for example free radicals) or a means such as UV light. Some other means of gelling or polymerization include matragel, polyacrylamide, mixed polysaccharides, etc. Some example initiators can be temperature, UV irradiation, etc.
In a further example, one of the DNA primers used for amplification can be attached to one of the molecules that will form the polymerized matrix. Attachment can be through a direct chemical attachment, or through a secondary attachment such as biotin-streptavidin attachment. In this example, the DNA will become physically attached to the formed solid-phase that occurs after solidification of the droplet. Using one of several gelling or polymerizing methods it should be possible to further manipulate these droplets.
One could also either exchange or wash away unincorporated nucleotides, primers and exchange buffers so as to remove the initial amplification buffers, polymerases, etc. In an example of further droplet manipulation wherein one of the strands is polymerized from an attached DNA amplification primer, one could treat the polymerized or gelled droplet with a base solution to disassociate the two DNA strands and elute, from the gelled or polymerized droplet, the unattached strand.
For protein analysis, proteins can be either trapped within, or attached to the gel or polymer matrix. If attached, it can be through a covalent linkage or through an affinity tag, for example his6 or avi-tag. The proteins can be added to droplets containing gel or polymer reagent, or they can be formulated along with the gel or polymerization reagent. Variations that include both are also possible. The protein can be added to the droplets. Additionally, it is possible to add DNA to the droplet and allow in vitro transcription/translation to synthesize the protein.
The droplets can be kept in liquid form on the microfluidic device and either gelled or polymerized upon removal, or can be gelled or polymerized within the droplets anywhere on the device after the droplets have been formed.
In an example, multiple plasmids are formulated into a droplet along with an in vitro transcription/translation reaction. Genes, encoded by the plasmids, are translated and transcribed to protein molecules. The protein molecules attach to the polymer via an avi-tag, the droplets are allowed to gel and the plasmid molecules become ‘fixed’ or ‘trapped’ within the gel. The gelled droplets are collected, the emulsion is broken and the solidified droplets collected and washed. As an example application, DNA is amplified within a droplet wherein one primer is physically attached to a polymer monomer. The droplet is then combined with a droplet containing the enzymes DNA polymerase, luciferase and sulfurylase. The merged droplets are allowed to gel or polymerize, they are collected, and if needed, washed. These washed gelled droplets can then used for a DNA sequencing reaction.
Surfactants
The fluids used in the invention may contain one or more additives, such as agents which reduce surface tensions (surfactants). Surfactants can include Tween, Span, fluorosurfactants, and other agents that are soluble in oil relative to water. In some applications, performance is improved by adding a second surfactant to the aqueous phase. Surfactants can aid in controlling or optimizing droplet size, flow and uniformity, for example by reducing the shear force needed to extrude or inject droplets into an intersecting channel. This can affect droplet volume and periodicity, or the rate or frequency at which droplets break off into an intersecting channel. Furthermore, the surfactant can serve to stabilize aqueous emulsions in fluorinated oils from coalescing.
The droplets may be coated with a surfactant. Preferred surfactants that may be added to the continuous phase fluid include, but are not limited to, surfactants such as sorbitan-based carboxylic acid esters (e.g., the “Span” surfactants, Fluka Chemika), including sorbitan monolaurate (Span 20), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60) and sorbitan monooleate (Span 80), and perfluorinated polyethers (e.g., DuPont Krytox 157 FSL, FSM, and/or FSH). Other non-limiting examples of non-ionic surfactants which may be used include polyoxyethylenated alkylphenols (for example, nonyl-, p-dodecyl-, and dinonylphenols), polyoxyethylenated straight chain alcohols, polyoxyethylenated polyoxypropylene glycols, polyoxyethylenated mercaptans, long chair carboxylic acid esters (for example, glyceryl and polyglycerl esters of natural fatty acids, propylene glycol, sorbitol, polyoxyethylenated sorbitol esters, polyoxyethylene glycol esters, etc.) and alkanolamines (e.g., diethanolamine-fatty acid condensates and isopropanolamine-fatty acid condensates). In addition, ionic surfactants such as sodium dodecyl sulfate (SDS) may also be used. However, such surfactants are generally less preferably for many embodiments of the invention. For instance, in those embodiments where aqueous droplets are used as nanoreactors for chemical reactions (including biochemical reactions) or are used to analyze and/or sort biomaterials, a water soluble surfactant such as SDS may denature or inactivate the contents of the droplet.
The carrier fluid can be an oil (e.g., decane, tetradecane or hexadecane) or fluorocarbon oil that contains a surfactant (e.g., a non-ionic surfactant such as a Span surfactant) as an additive (preferably between about 0.2 and 5% by volume, more preferably about 2%). A user can preferably cause the carrier fluid to flow through channels of the microfluidic device so that the surfactant in the carrier fluid coats the channel walls.
In one embodiment, the fluorosurfactant can be prepared by reacting the perflourinated polyether DuPont Krytox 157 FSL, FSM, or FSH with aqueous ammonium hydroxide in a volatile fluorinated solvent. The solvent and residual water and ammonia can be removed with a rotary evaporator. The surfactant can then be dissolved (e.g., 2.5 wt %) in a fluorinated oil (e.g., Flourinert (3M)), which then serves as the continuous phase of the emulsion.
Driving Forces
The invention can use pressure drive flow control, e.g., utilizing valves and pumps, to manipulate the flow of cells, particles, molecules, enzymes or reagents in one or more directions and/or into one or more channels of a microfluidic device. However, other methods may also be used, alone or in combination with pumps and valves, such as electro-osmotic flow control, electrophoresis and dielectrophoresis (Fulwyer, Science 156, 910 (1974); Li and Harrison, Analytical Chemistry 69, 1564 (1997); Fiedler, et al. Analytical Chemistry 70, 1909-1915 (1998); U.S. Pat. No. 5,656,155). Application of these techniques according to the invention provides more rapid and accurate devices and methods for analysis or sorting, for example, because the sorting occurs at or in a sorting module that can be placed at or immediately after a detection module. This provides a shorter distance for molecules or cells to travel, they can move more rapidly and with less turbulence, and can more readily be moved, examined, and sorted in single file, i.e., one at a time.
Positive displacement pressure driven flow is a preferred way of controlling fluid flow and dielectrophoresis is a preferred way of manipulating droplets within that flow.
The pressure at the inlet module can also be regulated by adjusting the pressure on the main and sample inlet channels, for example, with pressurized syringes feeding into those inlet channels. By controlling the pressure difference between the oil and water sources at the inlet module, the size and periodicity of the droplets generated may be regulated. Alternatively, a valve may be placed at or coincident to either the inlet module or the sample inlet channel connected thereto to control the flow of solution into the inlet module, thereby controlling the size and periodicity of the droplets. Periodicity and droplet volume may also depend on channel diameter, the viscosity of the fluids, and shear pressure.
Without being bound by any theory, electro-osmosis is believed to produce motion in a stream containing ions e.g. a liquid such as a buffer, by application of a voltage differential or charge gradient between two or more electrodes. Neutral (uncharged) molecules or cells can be carried by the stream. Electro-osmosis is particularly suitable for rapidly changing the course, direction or speed of flow. Electrophoresis is believed to produce movement of charged objects in a fluid toward one or more electrodes of opposite charge, and away from one on or more electrodes of like charge. Where an aqueous phase is combined with an oil phase, aqueous droplets are encapsulated or separated from each other by oil. Typically, the oil phase is not an electrical conductor and may insulate the droplets from the electro-osmotic field. In this example, electro-osmosis may be used to drive the flow of droplets if the oil is modified to carry or react to an electrical field, or if the oil is substituted for another phase that is immiscible in water but which does not insulate the water phase from electrical fields.
Dielectrophoresis is believed to produce movement of dielectric objects, which have no net charge, but have regions that are positively or negatively charged in relation to each other. Alternating, non-homogeneous electric fields in the presence of droplets and/or particles, such as cells or molecules, cause the droplets and/or particles to become electrically polarized and thus to experience dielectrophoretic forces. Depending on the dielectric polarizability of the particles and the suspending medium, dielectric particles will move either toward the regions of high field strength or low field strength. For example, the polarizability of living cells depends on their composition, morphology, and phenotype and is highly dependent on the frequency of the applied electrical field. Thus, cells of different types and in different physiological states generally possess distinctly different dielectric properties, which may provide a basis for cell separation, e.g., by differential dielecrophoretic forces. Likewise, the polarizability of droplets also depends upon their size, shape and composition. For example, droplets that contain salts can be polarized. According to formulas provided in Fiedler, et al. Analytical Chemistry 70, 1909-1915 (1998), individual manipulation of sine, droplets requires field differences (inhomogeneities) with dimensions close to the droplets.
The term “dielectrophoretic force gradient” means a dielectrophoretic force is exerted on an object in an electric field provided that the object has a different dielectric constant than the surrounding media. This force can either pull the object into the region of larger field or push it out of the region of larger field. The force is attractive or repulsive depending respectively on whether the object or the surrounding media has the larger dielectric constant.
Manipulation is also dependent on permittivity (a dielectric property) of the droplets and/or particles with the suspending medium. Thus, polymer particles, living cells show negative—dielectrophoresis at high-field frequencies in water. For example, dielectrophoretic forces experienced by a latex sphere in a 0.5 MV/m field (10 V for a 20 micron electrode gap) in water are predicted to be about 0.2 piconewtons (pN) for a 3.4 micron latex sphere to 15 pN for a 15 micron latex sphere (Fiedler, et al. Analytical Chemistry 70, 1909-1915 (1998)). These values are mostly greater than the hydrodynamic forces experienced by the sphere in a stream (about 0.3 pN for a 3.4 micron sphere and 1.5 pN for a 15 micron sphere). Therefore, manipulation of individual cells or particles can be accomplished in a streaming fluid, such as in a cell sorter device, using dielectrophoresis. Using conventional semiconductor technologies, electrodes can be microfabricated onto a substrate to control the force fields in a microfabricated sorting device of the invention. Dielectrophoresis is particularly suitable for moving objects that are electrical conductors. The use of AC current is preferred, to prevent permanent alignment of ions. Megahertz frequencies are suitable to provide a net alignment, attractive force, and motion over relatively long distances. See U.S. Pat. No. 5,454,472.
Radiation pressure can also be used in the invention to deflect and move objects, e.g. droplets and particles (molecules, cells, particles, etc.) contained therein, with focused beams of light such as lasers. Flow can also be obtained and controlled by providing a pressure differential or gradient between one or more channels of a device or in a method of the invention.
Molecules, cells or particles (or droplets containing molecules, cells or particles) can be moved by direct mechanical switching, e.g., with on-off valves or by squeezing the channels. Pressure control may also be used, for example, by raising or lowering an output well to change the pressure inside the channels on the chip. See, e.g., the devices and methods described U.S. Pat. No. 6,540,895. These methods and devices can further be used in combination with the methods and devices described in pending U.S. Patent Application Publication No. 20010029983 and 20050226742. Different switching and flow control mechanisms can be combined on one chip or in one device and can work independently or together as desired.
Inlet Module
The microfluidic device of the present invention includes one or more inlet modules. An “inlet module” is an area of a microfluidic substrate device that receives molecules, cells, small molecules or particles for additional coalescence, detection and/or sorting. The inlet module can contain one or more inlet channels, wells or reservoirs, openings, and other features which facilitate the entry of molecules, cells, small molecules or particles into the substrate. A substrate may contain more than one inlet module if desired. Different sample inlet channels can communicate with the main channel at different inlet modules. Alternately, different sample inlet channels can communication with the main channel at the same inlet module. The inlet module is in fluid communication with the main channel. The inlet module generally comprises a junction between the sample inlet channel and the main channel such that a solution of a sample (i.e., a fluid containing a sample such as molecules, cells, small molecules (organic or inorganic) or particles) is introduced to the main channel and forms a plurality of droplets. The sample solution can be pressurized. The sample inlet channel can intersect the main channel such that the sample solution is introduced into the main channel at an angle perpendicular to a stream of fluid passing through the main channel. For example, the sample inlet channel and main channel intercept at a T-shaped junction; i.e., such that the sample inlet channel is perpendicular (90 degrees) to the main channel. However, the sample inlet channel can intercept the main channel at any angle, and need not introduce the sample fluid to the channel at an angle that is perpendicular to that flow. The angle between intersecting channels is in the range of from about 60 to about 120 degrees. Particular exemplary angles are 45, 60, 90, and 120 degrees.
Embodiments of the invention are also provided in which there are two or more inlet modules introducing droplets of samples into the main channel. For example, a first inlet module may introduce droplets of a first sample into a flow of fluid in the main channel and a second inlet module may introduce droplets of a second sample into the flow of fluid in main channel, and so forth. The second inlet module is preferably downstream from the first inlet module (e.g., about 30 μm). The fluids introduced into the two or more different inlet modules can comprise the same fluid or the same type of fluid (e.g., different aqueous solutions). For example, droplets of an aqueous solution containing an enzyme are introduced into the main channel at the first inlet module and droplets of aqueous solution containing a substrate for the enzyme are introduced into the main channel at the second inlet module. Alternatively, the droplets introduced at the different inlet modules may be droplets of different fluids which may be compatible or incompatible. For example, the different droplets may be different aqueous solutions, or droplets introduced at a first inlet module may be droplets of one fluid (e.g., an aqueous solution) whereas droplets introduced at a second inlet module may be another fluid (e.g., alcohol or oil).
Droplet Interdigitation
Particular design embodiments of the microfluidic device described herein allow for a more reproducible and controllable interdigitation of droplets of specific liquids followed by pair-wise coalescence of these droplets, described in further detail herein. The droplet pairs can contain liquids of different compositions and/or volumes, which would then combine to allow for a specific reaction to be investigated. The pair of droplets can come from any of the following: (i) two continuous aqueois streams and an oil stream; (ii) a continuous aqueous stream, an emulsion stream, and an oil stream, or (iii) two emulsion streams and an oil stream. The term “interdigitation” as used herein means pairing of droplets from separate aqueous streams, or from two separate inlet nozzles, for eventual coalescence.
The nozzle designs described herein enhance the interdigitation of droplets and further improves coalescence of droplets due to the better control of the interdigitation and smaller distance between pairs of droplets. The greater control over interdigitation allows for a perfect control over the frequency of either of the droplets. To obtain the optimum operation, the spacing between droplets and coupling of the droplets can be adjusted by adjusting flow of any of the streams, viscosity of the streams, nozzle design (including orifice diameter, the channel angle, and post-orifice neck of the nozzle
Reservoir/Well
A device of the invention can include a sample solution reservoir or well or other apparatus for introducing a sample to the device, at the inlet module, which is typically in fluid communication with inlet channel. Reservoirs and wells used for loading one or more samples onto the microfluidic device of the present invention, include but are not limited to, syringes, cartridges, vials, eppendorf tubes and cell culture materials (e.g., 96 well plates). A reservoir may facilitate introduction of molecules or cells into the device and into the sample inlet channel of each analysis unit.
Fluidic Interconnects
The microfluidic device can include a syringe (or other glass container) that is treated with a vapor or solution of an appropriate PEG-silane to effect the surface PEG functionalization. The purpose for treating the walls of glass containers (e.g., syringes) with a PEG functionality is to prevent biological adhesion to the inner walls of the container, which frustrates the proper transfer of biological/chemical materials into the microfluidic device of the present invention. The inlet channel is further connected to a means for introducing a sample to said device. The means can be a well or reservoir. The means can be temperature controlled. The inlet module may also contain a connector adapted to receive a suitable piece of tubing, such as liquid chromatography or HPLC tubing, through which a sample may be supplied. Such an arrangement facilitates introducing the sample solution under positive pressure in order to achieve a desired infusion rate at the inlet module.
The interconnections, including tubes, must be extremely clean and make excellent bonding with the PDMS surface in order to allow proper operation of the device. The difficulty in making a fluidic! connection to a microfluidic device is primarily due to the difficulty in transitioning from a macroscopic fluid line into the device while minimizing dead volume.
In order to minimize contamination and leakage and allow for greater reproducibility and reliability are improved, tubes and interconnects for the PDMS slab can be cured in place. The tubes and interconnects can be placed in position by applying a UV-cured adhesive to allow for holding the tubes in place on the silicone wafer. Once the tubes are placed in position, PDMS can be poured over the wafer and cured. The cured PDMS, along with the tubes in place, can be peeled off of the silicone wafer easily. This process can be applied to fluidics channels as well as other connection channels. Once the adhesive is applied onto the wafer, the process will allow for quick templating of PDMS slabs with exact reproducibility of channel locations and cleanliness. Tubes of any size can be implemented for this process. This process allows for less stress on the interconnection joints and smaller interconnection footprints in the device (see, for example, PCT/US2006/02186 filed on Jun. 1, 2006; PCT/US2006/021280 filed on Jun. 1, 2006 and PCT/US2006/021380 filed on Jun. 1, 2006, each of which is incorporated by reference in their entirety for all purposes).
The tubing side of the interconnect can be mounted into a retaining block that provides precise registration of the tubing, while the microfluidic device can be positioned accurately in a carrier that the retaining block would align and clamp to. The total dead volume associated with these designs would be critically dependent on how accurately the two mating surfaces could be positioned relative to each other. The maximum force required to maintain the seal would be limited by the exact shape and composition of the sealing materials as well as the rigidity and strength of the device itself. The shapes of the mating surfaces can be tailored to the minimal leakage potential, sealing force required, and potential for misalignment. By way of non-limiting example, the single ring indicated in can be replaced with a series of rings of appropriate cross-sectional shape.
Reservoirs and wells used for loading one or more samples onto the microfluidic device of the present invention, include but are not limited to, syringes, cartridges, vials, eppendorf tubes and cell culture materials (e.g., 96 well plates) as described above. One of the issues to be resolved in loading samples into the inlet channel at the inlet module of the substrate is the size difference between the loading means or injection means, e.g., capillary or HPLC tubing and the inlet channel. It is necessary to create an interconnect and loading method which limits leaks and minimizes dead volume and compliance problems. Several devices and methods described in further detail herein address and solve these art problems.
Self-Aligning Fluidic Interconnects
The present invention includes one or more inlet modules comprising self-aligning fluidic interconnects proximate to one or more inlet channels to improve the efficiency of sample loading and/or injection.
The present invention proposes the use of small interconnects based on creating a radial seal instead of a face seal between the microfluidic device and interconnect. The inserted interconnect would have a larger diameter than the mating feature on the device. When inserted, the stretching of the chip would provide the sealing force needed to make a leak-free seal between the external fluid lines and the microfluidic device. FIG. 11 details design possibilities for making this seal.
Studies were performed with the leftmost design of FIG. 1 using a cast hole in PDMS and 1/32″ PEEK tubing, which showed that the seal was able to withstand more than 90 PSI of pressure without leakage.
In order to handle instrument and chip manufacturing tolerances, the external interconnect must be self aligning and the “capture radius” of the molded hole must be large enough to reliably steer the interconnect to the sealing surfaces. FIG. 12 shows that the entrance to the molded hole is large enough to guarantee capture but tapers down to the sealing surfaces. The external interconnect could be made directly out of the tubing leading up to the microfluidic substrate, thus eliminating potential leak points and unswept volumes. As seen in FIG. 12 , the interconnect is surrounded by the substrate interconnects or “chip dock” for most of its length to make certain it is held within the tolerance stack-up of the system. The external interconnect is made from a hard but flexible material such as 1/32″ PEEK tubing. The features in the microfluidic device can be molded directly into it during the manufacturing process, while the inserted seals can be molded/machined directly onto the tubing ends or molded as individual pieces and mechanically fastened to the tubing. The retaining ferrule shown in FIG. 12 would be attached during manufacturing and provide good absolute referencing of the tube length. The ferrule could be an off-the-shelf component or a custom manufactured part and be made from, for example, a polymer, an elastomer, or a metal. The tubing end could be tapered on the end (top most diagram) or squared off (the figure above). The specific shape of the end will be controlled by how easily the microfluidic device will gall during insertion.
Alternatively, it is also possible to mold all the interconnects needed for each tube into a single monolithic self-aligned part as detailed in FIG. 13 . This may help reduce the difficulty in maintaining alignment of many external fluidic lines to the chip.
Methods for Molding Fluidic Interconnects Directly on the Substrate
The present invention also provides methods of direct molding of fluidic interconnects into a microfluidic device. Development of a commercial microfluidic platform requires a simple, reliable fluidic interconnect in order to reduce the chance of operator error and leaks. Molding these interconnects directly into the microfluidic device requires precise alignment of the molding pins to 30 the patterned shim (the “master” manufactured from Silicon/photoresist or made from some metal) used to form the microfluidic and electrical channels. The extreme tolerances required when molding with low viscosity elastomer such as PDMS requires near perfect sealing of the pin face to the mast r, while still accommodating imperfections in the master and assembly of the molding tool. In an embodiment, the present invention provides a precise and repeatable method of molding of interconnects while accommodating the imperfections in the molding process by introducing movable pins captured in an elastomeric sleeve molded directly into the tool. In order to effectively produce at relatively low volume and be able to inexpensively prototype devices, the tool must be able to use masters generated using standard photolithographic processes (e.g. silicon wafers patterned with SU
Figure US12337287-20250624-P00003
8).
FIG. 14 shows a schematic of a molding tool based on this concept. In FIG. 14 , the pins (orange) are captured within an elestomeric molded sleeve. A compression plate made from a rigid backer plate and foam rubber is used to apply gentle even pressure to the pins and generate the force needed to make the pins uniformly contact the master. The molded sleeve was found to be necessary to consistently prevent the uncured elastomer from penetrating the region between the pin and the top plate. Early designs used pins captured in tight clearance holes, and the pins would frequently bind in place (even with lubricant), preventing smooth motion of the pins and improper contact with the master. This would in turn cause a thin film of the elastomer to form between the bottom of the pin and the master (“Flash”). This flash prevents proper operation of the interconnects during chip operation. The addition of the elastomeric sleeves around each pin eliminated this problem, and produce consistent, reliable shutoff between the master and the pins.
Acoustic Actuator
The well or reservoir of the inlet module further include an acoustic actuator. To obtain one droplet comprising a single element of a specific biological/chemical material (e.g., a cell), separation of biological/chemical material, and uniformity of the number density of biological/chemical materials in a microfluidic channel is desirable. Accordingly, the microfluidic device can include an acoustic actuator. The loaded sample (biological/chemical material) can be well mixed and separated in a small chamber by acoustic wave before sending out to the nozzle region for encapsulation. The frequency of the acoustic wave should be fine tuned so as not to cause any damage to the cells. The biological effects of acoustic mixing have been well studied (e.g., in the ink-jet industry) and many published literatures also showed that piezoelectric microfluidic device can deliver intact biological payloads such as live microorganisms and DNA.
The design of the acoustic resonant can use a Piezoelectric bimorph flat plate located on the side of the carved resonant in the PDMS slab. The resonant inlet can connect to the cell flow input channel and the outlet can connect to the cell flow pinching channel. The piezoelectric driving way form can be carefully optimized to select the critical frequencies that can separate cells in fluids. There are five parameters to optimize beyond the frequency parameter and Lab electronics can be used to optimize the piezoelectric driving waveform. Afterwards, a low cost circuit can be designed to generate only the optimized waveform in a preferred microfluidic device.
Coalescence Module
The microfluidic device of the present invention also includes one or more coalescence modules. A “coalescence module” is within or coincident with at least a portion of the main channel at or downstream of the inlet module where molecules, cells, small molecules or particles comprised within droplets are brought within proximity of other droplets comprising molecules, cells, small molecules or particles and where the droplets in proximity fuse, coalesce or combine their contents. The coalescence module can also include an apparatus, for generating an electric force.
The electric force exerted on the fluidic droplet may be large enough to cause the droplet to move within the liquid. In some cases, the electric force exerted on the fluidic droplet may be used to direct a desired motion of the droplet within the liquid, for example, to or within a channel or a microfluidic channel (e.g., as further described herein), etc.
The electric field can be generated from an electric field ‘generator, i.e., a device or system able to create an electric field that can be applied to the fluid. The electric field generator may produce an AC field (i.e., one that varies periodically with respect to time, for example, sinusoidally, sawtooth, square, etc.), a DC field (i.e., one that is constant with respect to time), a pulsed field, etc. The electric field generator may be constructed and arranged to create an electric field within a fluid contained with a channel or a microfluidic channel. The electric field generator may be integral to or separate from the fluidic system containing the channel or microfluidic channel, according to some embodiments. As used herein, “integral” means that portions of the components integral to each other are joined in such a way that the components cannot be in manually separated from each other without cutting or breaking at least one of the components.
Techniques for producing a suitable electric field (which may be AC, DC, etc.) are known to those of ordinary skill in the art. For example, in one embodiment, an electric field is produced by applying voltage across a pair of electrodes, which may be positioned on or embedded within the fluidic system (for example, within a substrate defining the channel or microfluidic channel), and/or positioned proximate the fluid such that at least a portion of the electric field interacts with the fluid. The electrodes can be fashioned from any suitable electrode material or materials known to those of ordinary skill in the art, including, but not limited to, silver, gold, copper, carbon, platinum, copper, tungstein, tin, cadmium, nickel, indium tin oxide (“ITO”), etc., as well as combinations thereof.
Electrodes
The device can include channels for use in fluid control and other channels filled with a metal alloy for casting integrated metal alloy components (i.e., electrodes). Alternatively, the electrodes can be manufactured using other technologies (e.g., lithographically patterned electrodes made from indium tin oxide or a metal such as platinum). The microfluidic device can include metal alloy components useful for performing electrical functions on fluids, including but not limited to, coalescing droplets, charging droplets, sorting droplets, detecting droplets and shaking droplets to mix the contents of coalesced droplets. The device can contain more than one of the above mentioned components for more than one of the above mentioned functions.
The electrodes comprising metal alloy components may either terminate at fluid channels or be isolated from fluid channels. The electrodes can be constructed by filling the appropriate channels with metal alloy. One way this can be accomplished is to use positive pressure injection of the metal alloy in a melted state, such as with a syringe, into the channels, and then cool the metal alloy to a solid form. Another example is to use negative pressure to draw the metal alloy in a melted state into the channels, and then cool the metal alloy to a solid form. This can be accomplished for example by use of capillary forces. Another method of construction can use any of the above mentioned embodiments, and then flush out the metal alloy in a melted state with another liquid to define the geometry of the metal alloy components. Another example is to use any of the above mentioned embodiments, and then use a localized cold probe to define a solid termination point for the metal alloy, and then cool the remaining metal alloy to a solid form. A further example is to use another material, such as microscopic solder spheres or UV curable conductive ink, to form a barrier between fluid and metal alloy channels, to define the geometry of the metal alloy components.
The device can include a combination of both integrated metal alloy components and a patterned electrically conductive layer. The patterned electrically conductive layer can have features patterned such that their boundaries are within a leak-proof seal. The device can have a patterned electrically conductive feature as one of two charging electrodes and one integrated metal alloy component as the other of two charging electrodes.
The device can include a plurality of electrodes that are insulated from the fluid present in the device, and the method of operation including appropriate application of dielectrical signals and appropriate fluids. In known devices, the electrodes are typically in contact with the fluids in order to allow discharge of species that would otherwise screen the applied dielectric field. Whereas, in devices where the electrodes have been insulated from the fluid, this screening effect typically arises so quickly that the device is not useful for any significantly extended period of time. The drawbacks of electrodes in contact with the fluids vs. insulated electrodes are (a) degraded reliability against leaking (since the interface between the electrodes and the other components of the device may be more difficult to effect a leak-proof seal), and (b) degraded reliability against electrode corrosion (whose failure mode effects include failure of application of dielectric fields, and fluid channel contamination).
The device of the present invention comprising a plurality of electrodes that are insulated from the fluid present in the device counteracts this screening effect by extending the screening rise time and including a polarity switch for all of the different dielectric fields applied in the device. The screening rise time is extended by using fluids with dielectrical properties. A polarity switch for all of the different dielectric fields applied in the device is achieved by using an algorithm for dielectrical control, which switches the polarity of the dielectrical fields at a frequency sufficiently high to maintain proper dielectrical function of the device. This dielectrical control algorithm may also switch the polarity for the dielectric fields in a cascading, time controlled manner starting at the fluid origin point and progressing downstream, so that given fluid components experience one polarity at every point along their course. The device of the present invention can be used with metal alloy electrodes or using a combination of metal alloy electrodes and patterned conductive film electrodes.
The invention can provide a microfluidic device using injected electrodes. The interface between the microscopic electrode (typically 25 μm thick) and the macroscopic interconnect can easily fail if the joint between the two is flexed. The flexing of the joint can be eliminated by securing a firm material that serves to fasten, support, and re-enforce the joint (i.e., a grommet) into the interface. In order to prevent flexing, the mating surface of the device can be manufactured from a hard material such as glass or plastic. The electrical connection with the external system can be made by securing the device such that it connects to a spring loaded contact, which is either offset from the grommet (thereby minimizing the force applied to the solder region), or centered on the grommet (as long as the contact does not touch the solder).
The metal alloy components are also useful for performing optical functions on fluids, including but not limited to, optical detection of droplets in a geometry which may include a mirror.
To prevent leakage of fluid out of electrodes placed within microfluidic channels, the microfluidic device can include a layer patterned with channels for fluid control, and another layer with patterned electrically conductive features, where the features are patterned such that their boundaries are within a leak-proof seal. The leak-proof seal can be achieved at the interface between the unpatterned areas of the fluid control layer and the unpatterned areas of the electrically conductive layer. The leak-proof seal can also be achieved by a third interfacial layer between the fluid control layer and the unpatterned areas of the electrically conductive layer. The third interfacial layer can or can not be perforated at specific locations to allow contact between the fluid and the electrically conductive layer. Electrical access ports can also be patterned in the fluid control layer.
The electrodes and patterned electrically conductive layers as described can be associated with any module of the device (inlet module, coalescence module, mixing module, delay module, detection module and sorting module) to generate dielectric or electric forces to manipulate and control the droplets and their contents.
Effective control of uncharged droplets within microfluidic devices can require the generation of extremely strong dielectric field gradients. The fringe fields from the edges of a parallel plate capacitator can provide an excellent topology to form these gradients. The microfluidic device according to the present invention can include placing a fluidic channel between two parallel electrodes, which can result in a steep electric field gradient at the entrance to the electrodes due to edge effects at the ends of the electrode pair. Placing these pairs of electrodes at a symmetric channel split can allow precise bi-directional control of droplet within a device. Using the same principle, only with asymmetric splits, can allow single ended control of the droplet direction in the same manner. Alternatively, a variation on this geometry will allow precise control of the droplet phase by shifting.
In some cases, transparent or substantially transparent electrodes can be used. The electric field generator can be constructed and arranged (e.g., positioned) to create an electric field applicable to the fluid of at least about 0.01 V/micrometer, and, in some cases, at least about 0.03 V/micrometer, at least about 0.05 V/micrometer, at least about 0.08 V/micrometer, at least about 0.1 V/micrometer, at least about 0.3 V/micrometer, at least about 0.5 V/micrometer, at least about 0.7 V/micrometer, at least about 1 V/micrometer, at least about 1.2 V/micrometer, at least about 1.4 V/micrometer, at least about 1.6 V/micrometer, or at least about 2 V/micrometer. In some embodiments, even higher electric field intensities may be used, for example, at least about 2 V/micrometer, at least about 3 V/micrometer, at least about 5 V/micrometer, at least about 7 V/micrometer, or at least about 10 V/micrometer or more.
As described, an electric field may be applied to fluidic droplets to cause the droplets to experience an electric force. The electric force exerted on the fluidic droplets may be, in some cases, at least about 10−16N/micrometer3. In certain cases, the electric force exerted on the fluidic droplets may be greater, e.g., at least about 10−15 N/micrometer3, at least about 10−14 N/micrometer3, at least about 10−13N/micrometer3, at least about 10−12 N/micrometer3, at least about 10−11 N/micrometer3, at least about 10−10 N/micrometer3, at least about 10−9N/micrometer3, at least about 10−8 N/micrometer3, or at least about 10−7 N/micrometer3 or more. The electric force exerted on the fluidic droplets, relative to the surface area of the fluid, may be at least about 10−15N/micrometer2, and in some cases, at least about 10−14 N/micrometer2, at least about 10−13 N/micrometer2, at least about 10−12 N/micrometer2, at least about 10−11N/micrometer2, at least about 10−10 N/micrometer2, at least about 10−9 N/micrometer2, at least about 10−8 N/micrometer2, at least about 10−7 N/micrometer2, or at least about 10−6 N/micrometer2 or more. In yet other embodiments, the electric force exerted on the fluidic droplets may be at least about 10−9 N, at least about 10−8 N, at least about 10−7 N, at least about 10−6N, at least about 10−5 N, or at least about 10−4 N or more in some cases.
Channel Expansion Geometries
In preferred embodiments described herein, droplet coalescence is presently carried out by having two droplet forming nozzles emitting droplets into the same main channel. The size of the nozzles allow for one nozzle to form a large drop that fills the exhaust line while the other nozzle forms a drop that is smaller than the first. The smaller droplet is formed at a rate that is less than the larger droplet rate, which insures that at most one small droplet is between big droplets. Normally, the small droplet will catch up to the larger one over a relatively short distance, but sometimes the recirculation zone behind the large drop causes the small drop to separate from the large drop cyclically. In addition, the small drop occasionally does not catch up with the large one over the distance between the nozzles and the coalescing electrodes. Thus, in some situations is a need for a more robust coalescence scheme.
Geometric alterations in the coalescence module can create a more robust, reliable coalescence or fusing of droplets over a wider range of sizes and flows. The solution to improve the performance is to place an expansion in the main channel between the electrodes. FIG. 15 is a schematic diagram of the improved coalescence module. Optionally, a small constriction (neckdown) just before this expansion can be used to better align the droplets on their way into the coalescence point (also shown in the FIG. 15 ). This optional neckdown can help center the small droplet in the channel stream lines, reducing the chance that it will flow around the larger droplet prior to coalescing in the expansion. The electrode pair may be placed on either one side of the channel or on both sides.
The expansion in the coalescing region allows for a dramatic catching up of the small drop to the large drop, as shown through micrographs taken on an operating device. The volume of the expansion is big enough to slow the large droplet down so that the small drop always catches up to the large drop, but doesn't allow the next large drop to catch up and make contact with the pair to be coalesced. The electrodes allow for coalescence to take place when the drops are in contact with each other and passing through the field gradient.
Detection Module
The microfluidic device of the present invention can also include one or more detection modules. A “detection module” is a location within the device, typically within the main channel where molecules, cells, small molecules or particles are to be detected, identified, measured or interrogated on the basis of at least one predetermined characteristic. The molecules, cells, small molecules or particles can be examined one at a time, and the characteristic is detected or measured optically, for example, by testing for the presence or amount of a reporter. For example, the detection module is in communication with one or more detection apparatuses. The detection apparatuses can be optical or electrical detectors or combinations thereof. Examples of suitable detection apparatuses include optical waveguides, microscopes, diodes, light stimulating devices, (e.g., lasers), photo multiplier tubes, and processors (e.g., computers and software), and combinations thereof, which cooperate to detect a signal representative of a characteristic, marker, or reporter, and to determine and direct the measurement or the sorting action at the sorting module. However, other detection techniques can also be employed
The term “determining,” as used herein, generally refers to the analysis or measurement of a species, for example, quantitatively or qualitatively, and/or the detection of the presence or absence of species. “Determining” may also refer to the analysis or measurement of an interaction between more species, for example, quantitatively or qualitatively, or by detecting the presence or absence of the interaction. Examples of suitable techniques include, but are not limited to, spectroscopy such as infrared, absorption, fluorescence, UV/visible, FTIR (“Fourier Transform Infrared Spectroscopy”), or Raman; gravimetric techniques; ellipsometry; piezoelectric measurements; immunoassays; electrochemical measurements; optical measurements such as optical density measurements; circular dichroism; light scattering measurements such as quasielectric light scattering; polarimetry; refractometry; or turbidity measurements as described further herein.
A detection module is within, communicating or coincident with a portion of the main channel at or downstream of the inlet module and, in sorting embodiments, at, proximate to, or upstream of, the sorting module or branch point. The sorting module may be located immediately downstream of the detection module or it may be separated by a suitable distance consistent with the size of the molecules, the channel dimensions and the detection system. Precise boundaries for the detection module are not required, but are preferred.
Detection modules used for detecting molecules and cells have a cross-sectional area large enough to allow a desired molecule, cells, bead, or particles to pass through without being substantially slowed down relative to the flow carrying it. The dimensions of the detection module are influenced by the nature of the sample under study and, in particular, by the size of the droplets, beads. particles, molecules or cells (including virions) under study. For example, mammalian cells can have a diameter of about 1 to 50 microns, more typically 10 to 30 microns, although some mammalian cells (e.g., fat cells) can be larger than 120 microns. Plant cells are generally 10 to 100 microns. However, other molecules or particles can be smaller with a diameter from about 20 nm to about 500 nm.
Waveguides
The present invention provides self-aligning optical waveguides and optical elements (lenses, mirrors, interconnects, etc.) for detection and control of droplets. Such waveguides can be provide well defined optical access to the fluidic channels to permit optical scattering, absorption, fluorescence, or any other optical measurement technique.
In order to create the waveguides, a separate series of channels and useful shapes (lenses, mirrors, etc) can be created either simultaneously within the other channels in the substrate (i.e. in the same processing step) or in successive steps. The reusable master created in this way can then used to form the waveguide components and fluid channels without the need for special fixturing or careful shipment in subsequent steps. The extra channels or shapes can then filled with a high index of refraction liquid (for waveguides) or reflective material (for mirrors) through injection into the channel or void. The liquid can either remain as a fluid or be allowed to solidify. UV cure epoxies used by the telecommunications industry are excellent choices for the waveguide materials. Possible waveguide geometry can include a focusing lens and a back-reflecting mirror.
Sensors
One or more detections sensors and/or processors may be positioned to be in sensing communication with the fluidic droplet. “Sensing communication,” as used herein, means that the sensor may be positioned anywhere such that the fluidic droplet within the fluidic system (e.g., within a channel), and/or a portion of the fluidic system containing the fluidic droplet may be sensed and/or determined in some fashion. For example, the sensor may be in sensing communication with the fluidic droplet and/or the portion of the fluidic system containing the fluidic droplet fluidly, optically or visually, thermally, pneumatically, electronically, or the like. The sensor can be positioned proximate the fluidic system, for example, embedded within or integrally connected to a wall of a channel, or positioned separately from the fluidic system but with physical, electrical, and/or optical communication with the fluidic system so as to be able to sense and/or determine the fluidic droplet and/or a portion of the fluidic system containing the fluidic droplet (e.g., a channel or a microchannel, a liquid containing the fluidic droplet, etc.). For example, a sensor may be free of any physical connection with a channel containing a droplet, but may be positioned so as to detect electromagnetic radiation arising from the droplet or the fluidic system, such as infrared, ultraviolet, or visible light. The electromagnetic radiation may be produced by the droplet, and/or may arise from other portions of the fluidic system (or externally of the fluidic system) and interact with the fluidic droplet and/or the portion of the fluidic system containing the fluidic droplet in such as a manner as to indicate one or more characteristics of the fluidic droplet, for example, through absorption, reflection, diffraction, refraction, fluorescence, phosphorescence, changes in polarity, phase changes, changes with respect to time, etc. As an example, a laser may be directed towards the fluidic droplet and/or the liquid surrounding the fluidic droplet, and the fluorescence of the fluidic droplet and/or the surrounding liquid may be determined. “Sensing communication,” as used herein may also be direct or indirect. As an example, light from the fluidic droplet may be directed to a sensor, or directed first through a fiber optic system, a waveguide, etc., before being directed to a sensor.
Non-limiting examples of detection sensors useful in the invention include optical or electromagnetically-based systems. For example, the sensor may be a fluorescence sensor (e.g., stimulated by a laser), a microscopy system (which may include a camera or other recording device), or the like. As another example, the sensor may be an electronic sensor, e.g., a sensor able to determine an electric field or other electrical characteristic. For example, the sensor may detect capacitance, inductance, etc., of a fluidic droplet and/or the portion of the fluidic system containing the fluidic droplet. In some cases, the sensor may be connected to a processor, which in turn, cause an operation to be performed on the fluidic droplet, for example, by sorting the droplet.
Characteristics
Characteristics determinable with respect to the droplet and usable in the invention can be identified by those of ordinary skill in the art. Non-limiting examples of such characteristics include fluorescence, spectroscopy (e.g., optical, infrared, ultraviolet, etc.), radioactivity, mass, volume, density, temperature, viscosity, pH, concentration of a substance, such as a biological substance (e.g., a protein, a nucleic acid, etc.), or the like.
A corresponding signal is then produced, for example indicating that “yes” the characteristic is present, or “no” it is not. The signal may correspond to a characteristic qualitatively or quantitatively. That is, the amount of the signal can be measured and can correspond to the degree to which a characteristic is present. For example, the strength of the signal may indicate the size of a molecule, or the potency or amount of an enzyme expressed by a cell, or a positive or negative reaction such as binding or hybridization of one molecule to another, or a chemical reaction of a substrate catalyzed by an enzyme. In response to the signal, data can be collected and/or a control system in the sorting module, if present, can be activated to divert a droplet into one branch channel or another for delivery to the collection module or waste module. Thus, in sorting embodiments, molecules or cells within a droplet at a sorting module can be sorted into an appropriate branch channel according to a signal produced by the corresponding examination at a detection module. The means of changing the flow path can be accomplished through mechanical, electrical, optical, or some other technique as described herein.
A preferred detector is an optical detector, such as a microscope, which may be coupled with a computer and/or other image processing or enhancement devices to process images or information produced by the microscope using known techniques. For example, molecules can be analyzed and/or sorted by size or molecular weight. Enzymes can be analyzed and/or sorted by the extent to which they catalyze chemical reaction of a substrate (conversely, substrate can be analyzed and/or sorted by the level of chemical reactivity catalyzed by an enzyme). Cells can be sorted according to whether they contain or produce a particular protein, by using an optical detector to examine each cell for an optic indication of the presence or amount of that protein. The protein may itself be detectable, for example by a characteristic fluorescence, or it may be labeled or associated with a reporter that produces a detectable signal when the desired protein is present, or is present in at least a threshold amount. There is no limit to the kind or number of characteristics that can be identified or measured using the techniques of the invention, which include without limitation surface characteristics of the cell and intracellular characteristics, provided only that the characteristic or characteristics of interest for sorting can be sufficiently identified and detected or measured to distinguish cells having the desired characteristic(s) from those which do not. For example, any label or reporter as described herein can be used as the basis for analyzing and/or sorting molecules or cells, i.e. detecting molecules or cells to be collected.
Fluorescence Polarization
As described herein, the biological/chemical entity to be analyzed may itself be detectable, for example by a characteristic fluorescence, or it may be labeled or associated with a reporter that produces a detectable signal when the desired protein is present, or is present in at least a threshold amount.
Luminescent colloidal semiconductor nanocrystals called quantum dots or q-dots (QD) are inorganic fluorophores that have the potential to circumvent some of the functional limitations encountered by organic dyes. In particular, CdSe—ZnS core-shell QDs exhibit size-dependent tunable photoluminescence (PL) with narrow emission bandwidths (FWHM˜30 to 45 nm) that span the visible spectrum and broad absorption bands. These allow simultaneous excitation of several particle sizes (colors) at a common wavelength. This, in turn, allows simultaneous resolution of several colors using standard instrumentation. CdSe—ZnS QDs also have high quantum yields, are resistant to photodegradation, and can be detected optically at concentrations comparable to organic dyes.
Quantum dots are nano-scale semiconductors typically consisting of materials such as crystalline cadmium selenide. The term ‘q-dot’ emphasizes the quantum confinement effect of these materials, and typically refers to fluorescent nanocrystals in the quantum confined size range. Quantum confinement refers to the light emission from bulk (macroscopic) semiconductors such as LEDs which results from exciting the semiconductor either electrically or by shining light on it, creating electron-hole pairs which, when they recombine, emit light. The energy, and therefore the wavelength, of the emitted light is governed by the composition of the semiconductor material. If, however, the physical size of the semiconductor is considerably reduced to be much smaller than the natural radius of the electron-hole pair (Bohr radius), additional energy is required to “confine” this excitation within the nanoscopic semiconductor structure leading to a shift in the emission to shorter wavelengths. Three different q-dots in several concentrations each can be placed in a microdroplet, and can then be used with a microfluidic device to decode what is in the drop. The Q-dot readout extension to the fluorescence station can be incorporated into the design of the microfluidic device. A series of dichroic beamsplitters, emission filters, and detectors can be stacked onto the system, allowing measurement of the required five emission channels (two fluorescence polarization signals and three q-dot bands).
Fluorescence Polarization (FP) detection technology enables homogeneous assays suitable for high throughput screening assays in the Drug Discovery field. The most common label in the assays is fluorescein. In FP-assay the fluorophore is excited with polarized light. Only fluorophores parallel to the light absorb and are excited. The excited state has a lifetime before the light emission occurs. this time the labeled fluorophore molecule rotates and the polarization of the light emitted differs from the excitation plane. To evaluate the polarization two measurements are needed: the first using a polarized emission filter parallel to the excitation filter (S-plane) and the second with a polarized emission filter perpendicular to the excitation filter (P-plane). The Fluorescence Polarization response is given as mP (milli-Polarization level) and is obtained from the equation:
Polarization(mP)=1000*(S−G*P)/(S+G*P)
Where S and P are background subtracted fluorescence count rates and G (grating) is an instrument and assay dependent factor.
The rotational speed of a molecule is dependent on the size of the molecule, temperature and viscosity of the solution. Fluorescein has a fluorescence lifetime suitable for the rotation speeds of molecules in bio-affinity assays like receptor-ligand binding assays or immunoassays of haptens. The basic principle is that the labeled compound is small and rotates rapidly (low polarization). When the labeled compound binds to the larger molecule, its rotation slows down considerably (polarization changes from low to high polarization). Thus, FP provides a direct readout of the extent of tracer binding to protein, nucleic acids, and other biopolymers.
Fluorescence polarization technology has been used in basic research and commercial diagnostic assays for many decades, but has begun to be widely used in drug discovery only in the past six years. Originally, FP assays for drug discovery were developed for single-tube analytical instruments, but the technology was rapidly converted to high-throughput screening assays when commercial plate readers with equivalent sensitivity became available. These assays include such well-known pharmaceutical targets such as kinases, phosphatases, proteases, G-protein coupled receptors, and nuclear receptors. Other homogeneous technologies based on fluorescence intensity have been developed. These include energy transfer, quenching, and enhancement assays. FP offers several advantages over these. The assays are usually easier to construct, since the tracers do not have to respond to binding by intensity changes. In addition, only one tracer is required and crude receptor preparations may be utilized. Furthermore, since FP is independent of intensity, it is relatively immune to colored solutions and cloudy suspensions. FP offers several advantages in the area of instrumentation. Because FP is a fundamental property of the molecule, and the reagents are stable, little or no standardization is required. FP is relatively insensitive to drift in detector gain settings and laser power.
The dyes chosen for FP are commonly used in most cell- and enzyme-based assays and are designated not to overlap significantly with the q-dots. The dyes are evaluated both independently and together with the q-dots (at first off-instrument) to assess the cross-talk. Preferably, the liquid q-dot labels are read outside a spectral wavelength band currently used in FACS analysis and sorting (i.e., the dyes flourescein, Cy3, Cy5, etc). This permits the use of currently-available assays (dependent on these dyes). Using specific q-dots, crosstalk is minimized.
Accordingly, the present invention provides methods to label droplets and/or nanoreactors formed on a microfluidic device by using only a single dye code to avoid cross-talk with other dyes during FP. Additionally, the present invention provides methods to create FP dye codes to label compounds contained within liquids (including droplets and/or nanoreactors) where the compound is designed to be differentiated by FP on a microfluidic device. In this manner, dye codes having the same color, absorption, and emission could be used to label compounds within liquids.
In one aspect, the present invention is directed to the use of fluorescence polarization to label liquids. Droplets can be labeled using several means. These labeling means include, but are not limited to, the use of different dyes, quantum dots, capacitance, opacity, light scattering, fluorecence intensity (FI), fluorescence lifetime (FL), fluorescence polarization (FP), circular dichroism (CD), fluorescence correlation and combinations of all of these previous labeling means. The following disclosure describes the use of FP and FI as a means to label droplets on a microfluidic device. In addition, the use of FL as a means to adjust the overall FP of a solution, and by varying the concentration of the total FI, to create a 2-dimensional encoding scheme is demonstrated.
In general, molecules that take up more volume will tumble slower than a smaller molecule coupled to the same fluorophore (see FIG. 16 ). FP is independent of the concentration of the dye; liquids can have vastly different concentrations of FITC in them yet still have identical FP measurements.
In a preferred embodiment, a FP dye is an organic dye that does not interfere with the assay dye is used. Furthermore, since the total intensity of the FP dye can be quantified, a second dimension in which to label the droplet is provided. Thus, one can exploit the differences in FP to create an encoding scheme of dye within a liquid solution, including droplets. An example is shown in FIG. 17 whereby the droplets are labeled with 3 differently-sized FITC molecules (i.e., three different droplets contain FITC molecules and FITC coupled to either biotin or streptavidin, respectively). Therefore, in a single dimension, FP can be used to create an encoding scheme. However, the present invention can also use Fluorescence Intensity (FI) of the overall solution to create even more labels in a second dimension. An example of labeling droplets in 2 dimensions is shown in FIG. 18 .
Interestingly, the differences of the fluorescence lifetime (FL) of two dyes with spectral overlap in the detected emission wavelength to change the overall FP of the combined solution can also be exploited (see FIGS. 18 and 19 ).
Although FIG. 17 discusses the use of multiple compounds to which a dye molecule is attached to span a range of FP, it is also possible to span the range using a high and low molecular weight compound set. As exemplified by FIG. 19 , a dye can be attached to a large compound (for example streptavidin) and kept at a fixed concentration, to which a smaller compound (for example, a free dye molecule) would be titrated into the same solution. The FP of the solution can be adjusted to be in discernable increments from the value of the large molecule to somewhere slightly greater than the FP of the smaller molecule. The [total] dye intensity can be varied by varying the concentration of the mixture of the two dye-attached compounds. By varying total dye concentration and the FP, two dimensions can be used to generate the FP dye codes (FPcodes). Accordingly, many FPcodes can be generated using only two compounds.
This could also include use of large fluorescent proteins such as GFP and the phycobiliproteins combined with a smaller molecule.
Examples of dyes commonly used in biological dyes are listed in the table below.
Examples
Excitation Wavelength Emission Wavelength of Compatible Dyes
450 500 Cyan 500
483 533 SYBR Green, FAM
523 568 HEX, VIC
558 610 RED 610
615 640 RED 640
650 670 CY5
In another aspect, the present invention is directed labeling solids using properties other than dye emission and dye concentration. In one embodiment the solid can include, for example, a bead or location on a solid support or chip. As demonstrated above for liquids, FI and FL can be two of many dimensions of characteristics used as labels. By way of non-limiting example, it is possible to use two dyes with different FL to change the overall FP for a solid such as a bead or other mobile solid support.
In another embodiment, a linker can be used to couple the dye to the bead. The linker can be varied so as to allow the dye to have differing degrees of freedom in which to rotate (i.e., tumble). Varying the linker in this manner can change the FP of the attached dye, which in unique combinations can be used as a label. In some embodiments, the beads can be swollen in organic solvent and the dyes held in place by hydrophobic forces. In this case, the FP, FI, FL methods described above for liquid labeling can also be used as a means for labeling the beads. A quenching molecule can also be used to change the characteristics of a dye. Such quenching can be continuous or brought about through the interaction of a molecule, such as a peptide or nucleic acid linker, with differing means of bringing molecules together depending on the strength of linker-internal interaction (e.g., a nucleotide stem loop structure of varying lengths).
The reactions analyzed on the virtual, random and non-random arrays (discussed briefly below) can be also increased beyond the two (cy3 and cy5 intensities) commonly used for multiplexing. For example, different FP, FI, etc can be used as a read-out.
Random array decoding: Beads of the prior art use one or more pre-attached oligonucleotide-coupled beads that are held in place in a fiber-optic faceplate (for example, those used by Illumina). The oligos on the beads are decoded using sequential hybridization of a labeled complementary oligo. The assay of the prior art uses a separate oligonucleotide complementary zipcode (‘Illumacode’) attached to each type of bead.
The invention described herein is superior to the methods of the prior art in that the FP, FI, FL-labeled bead or mobile solid support can be placed into a random array (e.g., a chip as manufactured by Illumina) and the FP, FI, FL used to decode the bead. The FP, FI, FL of the bead can be decoded before using the chip and the different beads ‘mapped’ as to their specific locations. Alternatively, the bead can be decoded during attachment of the assay read-out. Significantly, the methods described by the present invention can be used to pre-determine the location of each bead-type either before, or during analysis.
Virtual array decoding: Methods of the prior art use 2 lasers and 3 detectors to differentiate a set of 100 bead-types. The beads-types are differentiated by the FI of two different dyes Present in 1 of 10 concentrations (per dye) contained within the bead, and the assay detector is used to measure fluorescein concentration on the bead. The dyes, which are added to organic-solvent swollen beads, are not directly attached to the beads, but remain held within the bead by hydrophobic forces.
Using the methods of the present invention as described herein, a second detector to the machines of the prior art used to measure FP can be added, thereby adding a third dimension and extending the encoding scheme beyond the 100 available in the prior art.
Non-random array decoding: In chips of the prior art (such as those used by Affymetrix) oligonucleotides are synthesized directly on the chip. Decoding is simply a matter of knowing the location of the assay on the chip.
The methods as described herein can be advantageously used in conjunction with such chips to increase the number of things that can be simultaneously analyzed (i.e., multiplexed) on the chip. By way of non-limiting example, Cy3, Cy5, FL and FP can be used as analysis markers for hybridization reactions.
The present invention also provides methods for labeling micro or nano-sized droplets using Radio Frequency Identification (RFID). RFID tags can improve the identification of the contents within the droplets. Preferably, the droplets are utilized within a microfluidic device.
RFID is an automatic identification method, relying on storing and remotely retrieving data using devices called RFID tags or transponders. An RFID tag is an object that can be attached to or incorporated into a product, animal, or person for the purpose of identification using radio waves. Chip-based RFID tags contain silicon chips and antennae. Passive tags require no internal power source, whereas active tags require a power source. Hitachi has “powder” 0.05 mm×0.05 mm RFID chips. The new chips are 64 times smaller than the previous record holder, the 0.4 mm×0.4 mm mu-chips, and nine times smaller than Hitachi's last year prototype, and have room for a 128-bit ROM that can store a unique 38-digit ID number.
In one embodiment, a solution containing RFID tags are emulsified into droplets and are used as a label for the identification of the material within the droplet solution. Applications include, but are not limited to; genetics, genomics, proteomics, chemical synthesis, biofuels, and others.
Lasers
To detect a reporter or determine whether a molecule, cell or particle has a desired characteristic, the detection module may include an apparatus for stimulating a reporter for that characteristic to emit measurable light energy, e.g., a light source such as a laser, laser diode, light emitting diode (LED), high-intensity lamp, (e.g., mercury lamp), and the like. Where a lamp is used, the channels are preferably shielded from light in all regions except the detection module. Where a laser is used, the laser can be set to scan across a set of detection modules from different analysis units. In addition, laser diodes or LED's may be microfabricated into the same chip that contains the analysis units. Alternatively, laser diodes or LED's may be incorporated into a second chip (i.e., a laser diode chip) that is placed adjacent to the analysis or microchip such that the laser light from the diodes shines on the detection module(s).
An integrated semiconductor laser and/or an integrated photodiode detector can be included on the substrate in the vicinity of the detection module. This design provides the advantages of compactness and a shorter optical path for exciting and/or emitted radiation, thus minimizing distortion and losses.
Fluorescence produced by a reporter is excited using a laser beam focused on molecules (e.g., DNA, protein, enzyme or substrate) or cells passing through a detection region. Fluorescent reporters can include, but are not limited to, rhodamine, fluorescein, Texas red, Cy 3, Cy 5, phycobiliprotein (e.g., phycoerythrin), green fluorescent protein (GFP), YOYO-1 and PicoGreen. In molecular fingerprinting applications, the reporter labels can be fluorescently labeled single nucleotides, such as fluorescein-dNTP, rhodamine-dNTP, Cy3-dNTP, etc.; where dNTP represents dATP, dTTP, dUTP or dCTP. The reporter can also be chemically-modified single nucleotides, such as biotin-dNTP. The reporter can be fluorescently or chemically labeled amino acids or antibodies (which bind to a particular antigen, or fragment thereof, when expressed or displayed by a cell or virus).
The device can analyze and/or sort cells based on the level of expression of selected cell markers, such as cell surface markers, which have a detectable reporter bound thereto, in a manner similar to that currently employed using fluorescence-activated cell sorting (FACS) machines. Proteins or other characteristics within a cell, and which do not necessarily appear on the cell surface, can also be identified and used as a basis for sorting. The device can also determine the size or molecular weight of molecules such as polynucleotides or polypeptides (including enzymes and other proteins) or fragments thereof passing through the detection module. Alternatively, the device can determine the presence or degree of some other characteristic indicated by a reporter. If desired, the cells, particles or molecules can be sorted based on this analysis. The sorted cells, particles or molecules can be collected from the outlet channels in collection modules (or discarded in wasted modules) and used as needed. The collected cells, particles or molecules can be removed from the device or reintroduced to the device for additional coalescence, analysis and sorting.
Processors
As used herein, a “processor” or a “microprocessor” is any component or device able to receive a signal from one or more sensors, store the signal, and/or direct one or more responses (e.g., as described above), for example, by using a mathematical formula or an electronic or computational circuit. The signal may be any suitable signal indicative of the environmental factor determined by the sensor, for example a pneumatic signal, an electronic signal, an optical signal, a mechanical signal, etc.
The device of the present invention can comprise features, such as integrated metal alloy components and/or features patterned in an electrically conductive layer, for detecting droplets by broadcasting a signal around a droplet and picking up an electrical signal in proximity to the droplet.
Parallel Analysis
The droplet content detection can also be achieved by simultaneous detection of contents of multiple droplets in parallel using spectroscopic fluorescence imaging with sensitivity as high as single-molecule limit. One can spatially distribute droplets containing fluorescent entities such as fluorophore biological markers and/or quantum dots in a two-dimensional sheet in a microscopic field-of-view. The filed-of-view of those droplets can then be illuminated by a fluorescence excitation source and the resulting fluorescence can be spectroscopically imaged. Therefore, for a given fluorescence detection sensitivity, the throughput of fluorescence detection compared to a single-drop fluorescence detection method can be increased by a factor of a/b for a given sensitivity, where a is the number of droplets that can be imaged within a given field-of-view, and b is the ratio of the fluorescence sensitivity of a single-drop fluorescence detector compared to that of the multiple drop fluorescence detector. Furthermore, unlike single-drop fluorescent detection method where the drops are flowed through a detection volume so that their residence time in the detection volume, and hence the signal integration time and sensitivity, is limited, the residence time of the droplet in the field-of-view can be unlimited, thereby allowing sensitivity as high as the single-molecule limit.
Beads
The device of the present invention also comprises the use of beads and methods for analyzing and sorting beads (i.e, bead reader device). The device can read and either sort or not sort droplets containing one or more of a set of two or more beads. Each bead can be differentiated from each other bead within a set. Beads can be separated by several tags including, but not limited to, quantum dyes, fluorescent dyes, ratios of fluorescent dyes, radioactivity, radio-tags, etc. For example, a set of beads containing a ratio of two dyes in discrete amounts with an apparatus for detecting and differentiating beads containing one discrete ratio from the other beads in this set having a different ratio of the two dyes. The microfluidic device can include paramagnetic beads. The paramagnetic beads can introduce and remove chemical components from droplets using droplet coalescence and breakup events. The paramagnetic beads can also be used for sorting droplets.
The present invention provides methods of screening molecular libraries on beads through limited-dilusion-loading and then chemical or optical release inside of droplets. Provided are methods for chemical synthesis on a bead and releasing said chemical attached to the bead using a releasing means (chemical, UV light, heat, etc) within a droplet, and then combining a second droplet to the first droplet for further manipulation. For example, tea-bag synthesis of chemicals on a bead simultaneously with a means for identifying said bead (using, for example, a mass spec tag). Using the resulting mixed-chemistry beads in a droplet within a fluid flow, and exposing the beads to UV light to release the chemical synthesized from the bead into the droplet environment. Combining the droplet containing the released chemical with a droplet containing a cell, and performing a cell-based assay. Sorting droplets having the desired characteristics (for example, turn on of a reporter gene), and then analyzing the sorted beads using mass spectroscopy.
The device of the present invention can comprise column separation prior to bead sorting. A device containing a channel loaded with a separating means for chromatographically sorting the sample prior to droplet formation. Such separating means could include size, charge, hydrophobicity, atomic mass, etc. The separating can be done isocratic or by use of a means for generating a gradient chemically, (for example using salt or hydrophobicity), electrically, by pressure, or etc. For example, a channel is preloaded with Sepharose size exclusion media. A sample is loaded at one end, and the droplets are formed at an opposing end. The sample separates by size prior to becoming incorporated within a droplet.
Sorting Module
The microfluidic device of the present invention can further include one or more sorting modules. A “sorting module” is a junction of a channel where the flow of molecules, cells, small molecules or particles can change direction to enter one or more other channels, e.g., a branch channel for delivery to an outlet module (i.e., collection or waste module), depending on a signal received in connection with an examination in the detection module. Typically, a sorting module is monitored and/or under the control of a detection module, and therefore a sorting module may “correspond” to such detection module. The sorting region is in communication with and is influenced by one or more sorting apparatuses. A sorting apparatus comprises techniques or control systems, e.g., dielectric, electric, electro-osmotic, (micro-) valve, etc. A control system can employ a variety of sorting techniques to change or direct the flow of molecules, cells, small molecules or particles into a predetermined branch channel. A “branch channel” is a channel which is in communication with a sorting region and a main channel. The main channel can communicate with two or more branch channels at the sorting module or “branch point”, forming, for example, a T-shape or a Y-shape. Other shapes and channel geometries may be used as desired. Typically, a branch channel receives molecules, cells, small molecules or particles depending on the molecule, cells, small molecules or particles characteristic of interest as detected by the detection module and sorted at the sorting module. A branch channel can have an outlet module and/or terminate with a well or reservoir to allow collection or disposal (collection module or waste module, respectively) of the molecules, cells, small molecules or particles. Alternatively, a branch channel may be in communication with other channels to permit additional sorting.
The device of the present invention can further include one or more outlet modules. An “outlet module” is an area of the device that collects or dispenses molecules, cells, small molecules or particles after coalescence, detection and/or sorting. The outlet module can include a collection module and/or a waste module. The collection module can be connected to a means for storing a sample. The collection module can be a well or reservoir for collecting and containing droplets detected to have a specific predetermined characteristic in the detection module. The collection module can be temperature controlled. The waste module can be connected to a means for discarding a sample. The waste module can be a well or reservoir for collecting and containing droplets detected to not have a specific predetermined characteristic in the detection module. The outlet module is downstream from a sorting module, if present, or downstream from the detection model if a sorting module is not present. The outlet module may contain branch channels or outlet channels for connection to a collection module or waste module. A device can contain more than one outlet module.
A characteristic of a fluidic droplet may be sensed and/or determined in some fashion, for example, as described herein (e.g., fluorescence of the fluidic droplet may be determined), and, in response, an electric field may be applied or removed from the fluidic droplet to direct the fluidic droplet to a particular region (e.g. a channel). A fluidic droplet is preferably sorted or steered by inducing a dipole in the uncharged fluidic droplet (which may be initially charged or uncharged), and sorting or steering the droplet using an applied electric field. The electric field may be an AC field, a DC field, etc. For example, with reference to FIG. 20A, a channel 540, containing fluidic droplet 530 and liquid 535, divides into channel 542 and 544. Fluidic droplet 530 is uncharged. Electrode 526 is positioned near channel 542, while electrode 527 is positioned near channel 544. Electrode 528 is positioned near the junction of channels 540, 542, and 544. In FIGS. 20C and 20D, a dipole is induced in the fluidic droplet using electrodes 526, 527, and/or 528. In FIG. 20C, a dipole is induced in droplet 530 by applying an electric field 525 to the droplet using electrodes 527 and 528. Due to the strength of the electric field, the droplet is strongly attracted to the right, into channel 544. Similarly, in FIG. 20D, a dipole is induced in droplet 530 by applying an electric field 525 to the droplet using electrodes 526 and 528, causing the droplet to be attracted into channel 542. Thus, by applying the proper electric field, droplet 530 can be directed to either channel 542 or 544 as desired.
The present invention also provides improvements in the efficiency, accuracy, and reliability of the preferred dielectric droplet sorting technique described above. The single sided dielectric sorting relies on a combination of flow imbalance between the two exhaust legs and a switchable electric field to selectively sort out droplets of interest from the main sample stream. Sorting decisions are made based on some form of real time measurement of the droplet and its contents. FIGS. 21 and 22 depict many of the various possible fluid and electrode geometries possible for single sided dielectric sorting. FIG. 21 , Panels A-D show possible flow channel geometries that can be used in an asymmetric sorting application. Panel F, illustrates the use of a barrier, for example, a barrier where no fluid flow passes on its left side. Note these designs are only conceptual representation of the fluid channels, and actual designs may differ in absolute and relative dimensions as determined by one of ordinary skill in the art.
FIG. 22 shows the possible electrode geometries used in an asymmetric sorting application. Panel A shows the use of sharp tipped electrodes. Panel B shows broad tipped electrodes to increase the interaction time between the droplets and the electric field (the tips could be many drop diameters long). Panel C shows electrodes straddling the collection line. Panel D shows electrodes on opposite sides of the main channel. Panel E shows an Asymmetric Electrode Pair (the asymmetry may be present on any of the other electrode pair layouts as well). Note these designs are only conceptual representation of the electrodes, and actual designs may differ in absolute dimensions and electrode shape as determined by one of ordinary skill in the art. Although the fluid channel geometry is drawn as a “Y” junction, any of the channel geometries shown in FIG. 21 could be substituted in these drawings.
Typically, the flow rate of the collection leg is set to a value just below the level required to begin pulling droplets into the collection line (indicated as 40% in the figures, although the actual value may be differ from this and is dependent on the actual fluidic and electrode geometry, total flow, as well as droplet size and composition).
As an alternative design strategy, the collection leg can be operated at a flow rate at which the droplets would normally flow down the Sort collect line (i.e. change the flow splits shown in the diagrams from 40% collect/60% waste to 60% collect/40% waste), and keep the electric field energized until a droplet of interest is detected. At that time, the field would be briefly turned off, and the droplet would be pulled down the collection leg based on fluidic forces instead of electrical forces.
Alternately, a fluidic droplet may be directed by creating an electric charge (e.g., as previously described) on the droplet, and steering the droplet using an applied electric field, which may be an AC field, a DC field, etc. As an example, an electric field maybe selectively applied and removed (or a different electric field may be applied) as needed to direct the fluidic droplet to a particular region. The electric field may be selectively applied and removed as needed, in some embodiments, without substantially altering the flow of the liquid containing the fluidic droplet. For example, a liquid may flow on a substantially steady-state basis (i.e., the average flowrate of the liquid containing the fluidic droplet deviates by less than 20% or less than 15% of the steady-state flow or the expected value of the flow of liquid with respect to time, and in some cases, the average flowrate may deviate less than 10% or less than 5%) or other predetermined basis through a fluidic system of the invention (e.g., through a channel or a microchannel), and fluidic droplets contained within the liquid may be directed to various regions, e.g., using an electric field, without substantially altering the flow of the liquid through the fluidic system.
In other embodiments, however, the fluidic droplets may be screened or sorted within a fluidic system of the invention by altering the flow of the liquid containing the droplets. For instance, in one set of embodiments, a fluidic droplet may be steered or sorted by directing the liquid surrounding the fluidic droplet into a first channel, a second channel, etc.
In another set of embodiments, pressure within a fluidic system, for example, within different channels or within different portions of a channel, can be controlled to direct the flow of fluidic droplets. For example, a droplet can be directed toward a channel junction including multiple options for further direction of flow (e.g., directed toward a branch, or fork, in a channel defining optional downstream flow channels). Pressure within one or more of the optional downstream flow channels can be controlled to direct the droplet selectively into one of the channels, and changes in pressure can be effected on the order of the time required for successive droplets to reach the junction, such that the downstream flow path of each successive droplet can be independently controlled. In one arrangement, the expansion and/or contraction of liquid reservoirs may be used to steer or sort a fluidic droplet into a channel, e.g., by causing directed movement of the liquid containing the fluidic droplet. The liquid reservoirs may be positioned such that, when activated, the movement of liquid caused by the activated reservoirs causes the liquid to flow in a preferred direction, carrying the fluidic droplet in that preferred direction. For instance, the expansion of a liquid reservoir may cause a flow of liquid towards the reservoir, while the contraction of a liquid reservoir may cause a flow of liquid away from the reservoir. In some cases, the expansion and/or contraction of the liquid reservoir may be combined with other flow-controlling devices and methods, e.g., as described herein. Non limiting examples of devices able to cause the expansion and/or contraction of a liquid reservoir include pistons and piezoelectric components. In some cases, piezoelectric components may be particularly useful due to their relatively rapid response times, e.g., in response to an electrical signal.
In some embodiments, the fluidic droplets may be sorted into more than two channels. Alternately, a fluidic droplet may be sorted and/or split into two or more separate droplets, for example, depending on the particular application. Any of the above-described techniques may be used to spilt and/or sort droplets. As a non-limiting example, by applying (or removing) a first electric field to a device (or a portion thereof), a fluidic droplet may be directed to a first region or channel; by applying (or removing) a second electric field to the device (or a portion thereof), the droplet may be directed to a second region or channel; by applying a third electric field to the device (or a portion thereof), the droplet may be directed to a third region or channel; etc., where the electric fields may differ in some way, for example, in intensity, direction, frequency, duration, etc. In a series of droplets, each droplet may be independently sorted and/or split; for example, some droplets may be directed to one location or another, while other droplets may be split into multiple droplets directed to two or more locations.
In some cases, high sorting speeds may be achievable using certain systems and methods of the invention. For instance, at least about 1 droplet per second may be determined and/or sorted in some cases, and in other cases, at least about 10 droplets per second, at least about 20 droplets per second, at least about 30 droplets per second, at least about 100 droplets per second, at least about 200 droplets per second, at least about 300 droplets per second, at least about 500 droplets per second, at least about 750 droplets per second, at least about 1000 droplets per second, at least about 1500 droplets per second, at least about 2000 droplets per second, at least about 3000 droplets per second, at least about 5000 droplets per second, at least about 7500 droplets per second, at least about 10,000 droplets per second, at least about 15,000 droplets per second, at least about 20,000 droplets per second, at least about 30,000 droplets per second, at least about 50,000 droplets per second, at least about 75,000 droplets per second, at least about 100,000 droplets per second, at least about 150,000 droplets per second, at least about 200,000 droplets per second, at least about 300,000 droplets per second, at least about 500,000 droplets per second, at least about 750,000 droplets per second, at least about 1,000,000 droplets per second may be determined and/or sorted in such a fashion.
Multiple Measurement Sorting
In some embodiments, it may be useful to sort droplets based on two different measurements. For example, one might want to sort based on the ratio of two signals, sum of two signals, or difference between two signals. Specifically, this would be useful for cases when one would like to optimize an enzyme so that it work one substrate, but not another, or so that it works on two substrates. This is not easy to do using multiple rounds of selection on populations of droplets. To overcome this shortcoming of current sorting technology, the present invention provides a device comprising multiple channels with the appropriate geometry to split droplets, perform different experiments on the two daughter droplets and then reorder so that they pass sequential through the detector. The sums, ratios or differences in the two signals can then be calculated before the droplets enter the sorting bifurcation. An indicator dye or equivalent material may be added to one or both droplets to indicate when each droplet enters and leaves the laser. A representative sketch is shown in FIG. 23 .
Sample Recovery
The present invention proposes methods for recovering aqueous phase components from aqueous emulsions that have been collected on a microfluidic device in a minimum number of steps and in a gentle manner so as to minimize potential damage to cell viability.
In one aspect, a stable aqueous sample droplet emulsion containing aqueous phase components in a continuous phase carrier fluid is allowed to cream to the top of the continuous phase carrier oil. By way of nonlimiting example, the continuous phase carrier fluid can include perfluorocarbon oil that can have one or more stabilizing surfactants. The aqueous emulsion rises to the top or separates from the continuous phase carrier fluid by virtue of the density of the continuous phase fluid being greater than that of the aqueous phase emulsion. For example, the perfluorocarbon oil used in one embodiment of the device is 1.8, compared to the density of the aqueous emulsion, which is 1.0.
The creamed emulsion is then placed onto a second continuous phase carrier fluid which contains a de-stabilizing surfactant, such as a perfluorinated alcohol (e.g., 1H, 1H, 2H,2H-Perfluoro-1-octanol). The second continuous phase carrier fluid can also be a perfluorocarbon oil. Upon mixing, the aqueous emulsion begins to coalesce, and coalescence is completed by brief centrifugation at low speed (e.g., 1 minute at 2000 rpm in a microcentrifuge). The coalesced aqueous phase can now be removed (cells can be placed in an appropriate environment for further analysis).
Additional destabilizing surfactants and/or oil combinations can be identified or synthesized to be useful with this invention.
Mixing Module
The microfluidic device of the present invention can further include one or more mixing modules. Although coalescence of one or more droplets in one or more coalescence modules can be sufficient to mix the contents of the coalesced droplets (e.g., through rotating vortexes existing within the droplet), it should be noted that when two droplets fuse or coalesce, perfect mixing within the droplet does not instantaneously occur. Instead, for example, the coalesced droplet may initially be formed of a first fluid region (from the first droplet) and a second fluid region (from the second droplet). Thus, in some cases, the fluid regions may remain as separate regions, for example, due to internal “counter-revolutionary” flow within the fluidic droplet, thus resulting in a non-uniform fluidic droplet. A “mixing module” can comprise features for shaking or otherwise manipulate droplets so as to mix their contents. The mixing module is preferably downstream from the coalescing module and upstream from the detection module. The mixing module can include, but is not limited to, the use of channel geometries, acoustic actuators, metal alloy component electrodes or electrically conductive patterned electrodes to mix the contents of droplets and to reduce mixing times for fluids combined into a single droplet in the microfluidic device. For example, the fluidic droplet may be passed through one or more channels or other systems which cause the droplet to change its velocity and/or direction of movement. The change of direction may alter convection patterns within the droplet, causing the fluids to be at least partially mixed. Combinations are also possible.
For acoustic manipulation, the frequency of the acoustic wave should be fine tuned so as not to cause any damage to the cells. The biological effects of acoustic mixing have been well studied (e.g., in the ink-jet industry) and many published literatures also showed that piezoelectric microfluidic device can deliver intact biological payloads such as live microorganisms and DNA. In an example, the design of the acoustic resonant uses a Piezoelectric bimorph flat plate located on the side of the carved resonant in the PDMS slab. The piezoelectric driving waveform is carefully optimized to select the critical frequencies that can separate cells in fluids. There are five parameters to optimize beyond the frequency parameter. Lab electronics is used to optimize the piezoelectric driving waveform. Afterwards, a low cost circuit can be designed to generate only the optimized waveform in a preferred microfluidic device.
Other examples of fluidic mixing in droplets are described WO 2004/091763, incorporated herein by reference.
Delay Module
The microfluidic device of the present invention can further include one or more delay modules. The “delay module” can be a delay line. The operation of a microfluidics device where a reaction within a droplet is allowed to occur for a non-trivial length of time requires a delay line to increase the residence time within the device. For reactions demanding extensive residence time, longer or larger delay lines are required. Accordingly, the invention provides methods to increase residence times within microfluidic devices.
The delay module is in fluid communication with the main channel or it can be an elongated portion of the main channel itself. The delay module can be located downstream of the coalescence module and upstream of the detection module. The delay module can be a serpentine channel or a buoyant hourglass. The delay module can further comprise heating and cooling regions. The heating and cooling regions can be used for performing on-chip, flow-through PCR as further described herein.
The channel dimensions and configurations can be designed to accommodate the required residence time with minimum pressure drops across the device. For example, to accommodate very long delay lines within the microfluidic device, the device can comprise a multilayered PDMS slab which is composed of several patterned PDMS slabs.
The channel dimensions can also be designed so as to allow for required flow, residence time and pressure drop. Some channels may be required to be very large in width and height. In order to avoid collapse of the channels, the device includes support posts within the channel design. In order to reduce dead volume behind posts and further improve droplet stability, the support posts are designed to optimize a streamlined flow within the channel. These designs can include curved features as opposed to sharp edges.
To allow for longer period of device operation, delay lines can also be extended to the outside of the chip. The off-chip delay lines can be tubes within micron-sized internal diameter.
In order to allow more efficient use of available space and faster operation, in methods where droplets are charged, after charging, asymmetric splitting of oil and drops can be accommodated by siphoning off oil from channels after droplets are charged.
The delay lines can be in the form of a tower (i.e., a structure which is vertical with respect to the ambient gravitational field) as to allow buoyant forces to assist controlled droplet transport. Known delay lines involve transporting droplets by emulsifying them in a carrier fluid flowing in a channel and/or tube. Because the velocity profile of the carrier fluid through the cross-section of the channel and/or tube is not uniform, the velocity distribution of the droplets will not be narrow, which causes the delay time distribution of the droplets to not be narrow (i.e., some droplets will be delayed more or less than others).
The devices of the present invention can also include buoyancy-assisted microfluidic delay lines. In buoyancy-assisted microfluidic delay lines, buoyant forces act on droplets emulsified in a fluid in one or more towers. This can include allowing the tower to fill for the desired delay time, and then releasing the droplets. The tower can or can not continue to fill and release droplets as needed. In this example, one may desire to have a cylindrical tower section that is capped by a pyramidal funnel section. The tower can effectively functions as an hourglass. Droplets that have a density less than their carrier fluid are fed into the base of the tower, buoyantly rise to the top of the tower with a substantially uniform velocity distribution, and are funneled into a functional component of the microfluidic device (such as a y-branch). Carrier fluid is exhausted at the base of the tower at the same rate as it is introduced at the apex so that the net flow of carrier fluid through the delay line is zero. The tower and funnel sections can have any cross-sectional shape, such as circular, elliptical, or polygonal. The microfluidic device can include a tower with adjustable length.
The device can also include a switching network of twenty towers to guarantee a delay time dispersion of 5% (because 1/20=0.05). The capacity of each tower is 0.05*T, where T is the delay time. The concept includes, for example: (a) upon device start-up, filling the first tower for 0.05*T, but stop-cock its exhaust, and also have the other nineteen towers closed; (b) after 0.05*T, closing the first tower and filling the second between 0.05*T and 0.10*T; (c) repeating step (b) for the remaining eighteen towers; (d) at time T, allowing the first tower to exhaust; (e) at time 1.05*T, stop-cocking the exhaust of the first tower, allowing the second tower to exhaust, and allowing the first tower to fill; (f) at time 1.10*T, stop-cocking the exhaust of the second tower, allowing the third tower to exhaust, closing the first tower, and allowing the second tower to fill, and (g) repeating step (f) ad infinitum. More than twenty towers may provide an even tighter control over the width of the delay time dispersion. This scheme may require a valve network. This network of towers can be outside the microfluidic device.
The delay module can also include channels (e.g. the main channel) which has an altered geometry which permits the “parking” (e.g., slowing or stopping) of droplets within the microfluidic device.
In the methods provided herein, droplets are able to be parked in wells or channels at predefined locations. This can be done by creating discrete well-like indentions in the channel whereby a droplet ‘falls’ into the well and remains there as the fluid flows over it, or by using a technique entitled ‘by-pass pots’ whereby a droplet is used to block a small outlet in a well, thereby causing the flow to by-pass that droplet-containing well.
The instant invention is to use either of these techniques or any related technique, for example just stopping the drops in a channel, to position droplets at either random or predefined places within a microfluidics device. These random or predefined locations can then be queried at a later time-point for a reaction to have occurred, or for removal of the droplets using another means such as resuspension followed by aspiration.
In one example, a rolling circle amplification reaction is initiated in droplets, the droplets are then parked within the chip, and the amplification reaction allowed to proceed for a set period of time prior to stopping the reaction through the use of heat. The parked droplets are then dried in situ and the covering of the chip disassembled from the chip. One or a set of needle-like devices that are able to be lined up with the droplet parking space are then placed adjacent to or on top of the dried droplets and a liquid solution used to resuspend the material in the dried droplet that has been deposited into the chip, for further downstream processing.
In another example, to avoid possible diffusion of reactant contents from a first and a second set of reactions in droplets, the first reactions are created in 10 μm droplets, the droplets are dried within a channel parking space or by-pass pot which is able to hold a droplet of size larger than 10 μm, and the droplets are dried in situ. A second set of droplets that are larger than 10 μm are then allowed to proceed down said channel and when caught in said parking space or by-pass pot are able to resuspend the material from the first droplets that are dried along the walls of the first parking space or by-pass pot. In doing so, the second droplet is slightly larger than the first and that ensures that the material along the walls is ‘captured’ by the second droplet, and not allowed to diffuse away from the first droplet wall by diffusion. By doing so, use of surfactants becomes optional in either the first or second droplet formulations.
The instant invention also provides the following devices and methods for use in practicing the methods described herein. The PDMS substrate which comprises a portion of the microfluidic device can be covered or coated with an adhesive tape or strip that can removed by peeling. The PDMS substrate can also be bonded by an ultra thin silica that can be pierced by a set of needles. The silica may be spin coated or electro-plated onto a thin backing. Droplets can be dried onto a piece of paper such that can be detected by a second device to determine the Ncode within the droplet and to determine whether an amplification reaction has occurred within the droplet. A plate read comprising dried and undried spots using either an optical array device, such as found in high-end cameras or fiber, optic device is also contemplated. Dry Nitrogen can be utilized to dry the spots by either flowing it through the channel or placing the device into a dry-N2 chamber. Channels can be filled with dried nitrogen or salt run underneath or adjacent to the parking space channels to allow chemical or physical-type gradients to be set up in the chip. The channel walls can be coated with Steptavidin and the produced reactants, for example, DNA biotinylated so that it adheres in situ. Porous beads deposited into the wells can be used in combination with solutions without oils to wash the beads by flow, followed by re-depositing droplets with surfactants to recoat the beads. The wells within the substrate can be filled with many small beads by loading small beads into droplets, storing the droplets into individual wells containing apertures that are slightly smaller than the beads, breaking the droplets by drying or flow of aqueous solutions with or without surfactants into the channels and past the beads, and then re-encapsulating the beads in situ. A set of electrodes within or adjacent to the microfluidic substrate can be used to fuse two droplets in a storage/holding space. The electrodes may be perpendicular to the plane of the channels and either the electrodes or channels moved so as to allow droplet fusions to occur.
UV-Release Module
The microfluidic device of the present invention can further include one or more UV-release modules. The “UV-release module” is in fluid communication with the main channel. The UV release module is located downstream of the inlet module and upstream of the coalescence module. The UV-module can be a used in bead assays. Compounds from encapsulated beads can be cleaved in a UV-releasing module using UV light. Photolabile linkers can be broken down on demand after a single bead has been encapsulated thus releasing multiple copies of a single compound into solution. In the cell based assay disclosed herein the chemical compound assayed is desired to be in solution in order to penetrate the cell membrane. Furthermore, to ensure compartmentalization of a single, compound with a cell the cleavage of the compound from the solid support can only be done after the bead has been encapsulated. Photocleavable linkers can be utilized to cleave the compounds of the bead after drop formation by passing the drop through a UV-release module (i.e., laser of the appropriate wavelength).
The present invention also provides methods for chemical synthesis on a bead and releasing said chemical attached to the bead using a releasing means (chemical, UV light, heat, etc.) within a droplet, and then combining a second droplet to the first droplet for further manipulation. Preferably, the releasing means is a UV-module. For example, tea-bag synthesis of chemicals on a bead simultaneously with a means for identifying said bead (using, for example, a mass spec tag). Using the resulting mixed-chemistry beads in a droplet within a fluid flow, and exposing the beads to UV light to release the chemical synthesized from the bead into the droplet environment. Combining the droplet containing the released chemical with a droplet containing a cell, and performing a cell-based assay. Sorting droplets having the desired characteristics (for example, turn on of a reporter gene), and then analyzing the sorted beads using mass spectroscopy.
Kits
As a matter of convenience, predetermined amounts of the reagents, compound libraries, and/or emulsions described herein and employed in the present invention can be optionally provided in a kit in packaged combination to facilitate the application of the various assays and methods described herein. Such kits also typically include instructions for carrying out the subject assay, and may optionally include the fluid receptacle, e.g., the cuvette, multiwell plate, microfluidic device, etc. in which the reaction is to be carried out.
Typically, reagents included within the kit are uniquely labeled emulsions containing tissues, cells, particles, proteins, antibodies, amino acids, nucleotides, small molecules, substrates, and/or pharmaceuticals. These reagents may be provided in pre-measured container (e.g., vials or ampoules) which are co-packaged in a single box, pouch or the like that is ready for use. The container holding the reagents can be configured so as to readily attach to the fluid receptacle of the device in which the reaction is to be carried out (e.g., the inlet module of the microfluidic device as described herein). In one embodiment, the kit can include an RNAi kit. In another embodiment, the kit can include a chemical synthesis kit. It will be appreciated by persons of ordinary skill in the art that these embodiments are merely illustrative and that other kits are also within the scope of the present invention.
Methods
The microfluidic device of the present invention can be utilized to conduct numerous chemical and biological assays, including but not limited to, creating emulsion libraries, flow cytometry, gene amplification, isothermal gene amplification, DNA sequencing, SNP analysis, drug screening, RNAi analysis, karyotyping, creating microbial strains with improved biomass conversion, moving cells using optical tweezer/cell trapping, transformation of cells by electroporation, μTAS, and DNA hybridization.
Definitions
The terms used in this Specification generally have their ordinary meanings in the art, within the context of this invention and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the devices and methods of the invention and how to make and use them. It will be appreciated that the same thing can typically be described in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein. Synonyms for certain terms are provided. However, a recital of one or more synonyms does not exclude the use of other synonyms, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
The invention is also described by means of particular examples. However, the use of such examples anywhere in the specification, including examples of any terms discussed herein, is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described herein. Indeed, many modifications and variations of the invention will be apparent to those skilled in the art upon reading this specification and can be made without departing from its spirit and scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which the claims are entitled.
As used herein, “about” or “approximately” shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range.
The term “molecule” means any distinct or distinguishable structural unit of matter comprising one or more atoms, and includes for example polypeptides and polynucleotides.
The term “polymer” means any substance or compound that is composed of two or more building blocks (‘mers’) that are repetitively linked to each other. For example, a “dimer” is a compound in which two building blocks have been joined together.
The term “polynucleotide” as used herein refers to a polymeric molecule having a backbone that supports bases capable of hydrogen bonding to typical polynucleotides, where the polymer backbone presents the bases in a manner to permit such hydrogen bonding in a sequence specific fashion between the polymeric molecule and a typical polynucleotide (e.g., single-stranded DNA). Such bases are typically inosine, adenosine, guanosine, cytosine, uracil and thymidine. Polymeric molecules include double and single stranded RNA and DNA, and backbone modifications thereof, for example, methylphosphonate linkages.
Thus, a “polynucleotide” or “nucleotide sequence” is a series of nucleotide bases (also called “nucleotides”) generally in DNA and RNA, and means any chain of two or more nucleotides. A nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double or single stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and anti-sense polynucleotide (although only sense stands are being represented herein). This includes single- and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases, for example thio-uracil, thio-guanine and fluoro-uracil.
The polynucleotides herein may be flanked by natural regulatory sequences, or may be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5′- and 3′-non-coding regions, and the like. The nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Polynucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
The term “dielectrophoretic force gradient” means a dielectrophoretic force is exerted on an object in an electric field provided that the object has a different dielectric constant than the surrounding media. This force can either pull the object into the region of larger field or push it out of the region of larger field. The force is attractive or repulsive depending respectively on whether the object or the surrounding media has the larger dielectric constant.
“DNA” (deoxyribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and thymine (T), called nucleotide bases, that are linked together on a deoxyribose sugar backbone. DNA can have one strand of nucleotide bases, or two complimentary strands which may form a double helix structure. “RNA” (ribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and uracil (U), called nucleotide bases, that are linked together on a ribose sugar backbone. RNA typically has one strand of nucleotide bases.
A “polypeptide” (one or more peptides) is a chain of chemical building blocks called amino acids that are linked together by chemical bonds called peptide bonds. A “protein” is a polypeptide produced by a living organism. A protein or polypeptide may be “native” or “wild-type”, meaning that it occurs in nature; or it may be a “mutant”, “variant” or “modified”, meaning that it has been made, altered, derived, or is in some way different or changed from a native protein, or from another mutant.
An “enzyme” is a polypeptide molecule, usually a protein produced by a living organism, that catalyzes chemical reactions of other substances. The enzyme is not itself altered or destroyed upon completion of the reaction, and can therefore be used repeatedly to catalyze reactions. A “substrate” refers to any substance upon which an enzyme acts.
As used herein, “particles” means any substance that may be encapsulated within a droplet for analysis, reaction, sorting, or any operation according to the invention. Particles are not only objects such as microscopic beads (e.g., chromatographic and fluorescent beads), latex, glass, silica or paramagnetic beads, but also includes other encapsulating porous and/or biomaterials such as liposomes, vesicles and other emulsions. Beads ranging in size from 0.1 micron to 1 mm can be used in the devices and methods of the invention and are therefore encompassed with the term “particle” as used herein. The term particle also encompasses biological cells, as well as beads and other microscopic objects of similar size (e.g., from about 0.1 to 120 microns, and typically from about 1 to 50 microns) or smaller (e.g., from about 0.1 to 150 nm). The devices and methods of the invention are also directed to sorting and/or analyzing molecules of any kind, including polynucleotides, polypeptides and proteins (including enzymes) and their substrates and small molecules (organic or inorganic). Thus, the term particle further encompasses these materials.
The particles (including, e.g., cells and molecules) are sorted and/or analyzed by encapsulating the particles into individual droplets (e.g., droplets of aqueous solution in oil), and these droplets are then sorted, combined and/or analyzed in a microfabricated device. Accordingly, the term “droplet” generally includes anything that is or can be contained within a droplet.
A “small molecule” as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art.
As used herein, “cell” means any cell or cells, as well as viruses or any other particles having a microscopic size, e.g. a size that is similar to or smaller than that of a biological cell, and includes any prokaryotic or eukaryotic cell, e.g., bacteria, fungi, plant and animal cells. Cells are typically spherical, but can also be elongated, flattened, deformable and asymmetrical, i.e., non-spherical. The size or diameter of a cell typically ranges from about 0.1 to 120 microns, and typically is from about 1 to 50 microns. A cell may be living or dead. Since the microfabricated device of the invention is directed to sorting materials having a size similar to a biological cell (e.g. about 0.1 to 120 microns) or smaller (e.g., about 0.1 to 150 nm) any material having a size similar to or smaller than a biological cell can be characterized and sorted using the microfabricated device of the invention. Thus, the term cell shall further include microscopic beads (such as chromatographic and fluorescent beads), liposomes, emulsions, or any other encapsulating biomaterials and porous materials. Non-limiting examples include latex, glass, orparamagnetic beads; and vesicles such as emulsions and liposomes, and other porous materials such as silica beads. Beads ranging in size from 0.1 micron to 1 mm can also be used, for example in sorting a library of compounds produced by combinatorial chemistry. As used herein, a cell may be charged or uncharged. For example, charged beads may be used to facilitate flow or detection, or as a reporter. Biological cells, living or dead, may be charged for example by using a surfactant, such as SDS (sodium dodecyl sulfate). The term cell further encompasses “virions”, whether or not virions are expressly mentioned.
A “virion”, “virus particle” is the complete particle of a virus. Viruses typically comprise a nucleic acid core (comprising DNA or RNA) and, in certain viruses, a protein coat or “capsid” Certain viruses may have an outer protein covering called an “envelope”. A virion may be either living (i.e., “viable”) or dead (i.e., “non-viable”). A living or “viable” virus is one capable of infecting a living cell. Viruses are generally smaller than biological cells and typically range in size from about 20-25 nm diameter or less (parvoviridae, picornoviridae) to approximately 200-450 nm (poxviridae). However, some filamentous viruses may reach lengths of 2000 nm (closterviruses) and are therefore larger than some bacterial cells. Since the microfabricated device of the invention is particularly suited for sorting materials having a size similar to a virus (i.e., about 0.1 to 150 nm), any material having a size similar to a virion can be characterized and sorted using the microfabricated device of the invention. Non-limiting examples include latex, glass or paramagnetic beads; vesicles such as emulsions and liposomes; and other porous materials such as silica beads. Beads ranging in size from 0.1 to 150 nm can also be used, for example, in sorting a library of compounds produced by combinatorial chemistry. As used herein, a virion may be charged or uncharged. For example, charged beads may be used to facilitate flow or detection, or as a reporter. Biological viruses, whether viable or non-viable, may be charged, for example, by using a surfactant, such as SDS.
A “reporter” is any molecule, or a portion thereof, that is detectable, or measurable, for example, by optical detection. In addition, the reporter associates with a molecule, cell or virion or with a particular marker or characteristic of the molecule, cell or virion, or is itself detectable to permit identification of the molecule, cell or virion's, or the presence or absence of a characteristic of the molecule, cell or virion. In the case of molecules such as polynucleotides such characteristics include size, molecular weight, the presence or absence of particular constituents or moieties (such as particular nucleotide sequences or restrictions sites). In the case of cells, characteristics which may be marked by a reporter includes antibodies, proteins and sugar moieties, receptors, polynucleotides, and fragments thereof. The term “label” can be used interchangeably with “reporter”. The reporter is typically a dye, fluorescent, ultraviolet, or chemiluminescent agent, chromophore, or radio-label, any of which may be detected with or without some kind of stimulatory event, e.g., fluoresce with or without a reagent. In one embodiment, the reporter is a protein that is optically detectable without a device, e.g. a laser, to stimulate the reporter, such as horseradish peroxidase (HRP). A protein reporter can be expressed in the cell that is to be detected, and such expression may be indicative of the presence of the protein or it can indicate the presence of another protein that may or may not be coexpressed with the reporter. A reporter may also include any substance on or in a cell that causes a detectable reaction, for example by acting as a starting material, reactant or a catalyst for a reaction which produces a detectable product. Cells may be sorted, for example, based on the presence of the substance, or on the ability of the cell to produce the detectable product when the reporter substance is provided.
A “marker” is a characteristic of a molecule, cell or virion that is detectable or is made detectable by a reporter, or which may be coexpressed with a reporter. For molecules, a marker can be particular constituents or moieties, such as restrictions sites or particular nucleic acid sequences in the case of polynucleotides. For cells and virions, characteristics may include a protein, including enzyme, receptor and ligand proteins, saccharrides, polynucleotides, and combinations thereof, or any biological material associated with a cell or virion. The product of an ‘enzymatic reaction may also be used as a marker. The marker may be directly or indirectly associated with the reporter or can itself be a reporter. Thus, a marker is generally a distinguishing feature of a molecule, cell or virion, and a reporter is generally an agent which directly or indirectly identifies or permits measurement of a marker. These terms may, however, be used interchangeably.
The invention is further described below, by way of the following examples. The examples also illustrate useful methodology for practicing the invention. These examples do not limit the claimed invention.
EXAMPLES Example 1
The present invention provides methods for preparing a library of droplet emulsions, where each of the droplets is of the same, predetermined size (monodisperse). Further, present invention provides methods for deterministic lateral displacement for continuous particle separation, which can occur within droplets on a microfluidic device.
Particles in solution are usually separated according to size by exclusion or hydrodynamic chromatography. In the former, a sample mixture is injected at one end of a tube packed with porous beads and then washed through the tube. Particles smaller than the pore sizes enter the beads, which lengthen their migration path, and so they are on average eluted later than larger particles. Zones of particles of a given size broaden, however, because particles in each zone take many different paths leading to different retention times. This multipath effect reduces the resolution of size-exclusion chromatography. In hydrodynamic chromatography, a sample mixture is driven through a capillary by hydrodynamic flow, which has a parabolic flow profile. Large particles cannot intercept the low-velocity fluid near the capillary wall, and thus on average move faster and become separated from small particles. Multipath effects also limit the resolution of hydrodynamic chromatography, because each migration path samples different velocities in the parabolic flow.
Recently, Huang et al. Science 304(5673):987-90, 2004 and Davis et al Proc Natl Acad Sci USA. 103(40):14779-84, 2006 demonstrate a separation process that creates equivalent migration paths for each particle in a mixture, thereby eliminating multipath zone broadening. They describe a ‘lateral displacement’ means for separation of particles in solution based on particle size.
Lateral displacement means for sizing and separating droplets in solution (based on droplet size) can be utilized. The present invention relates to the generation of a microfluidic device consisting of raised pillars in both columns and rows that are designed for lateral diffusion. The pillars can be adjusted so as to be a means for separating droplets of similar sizes from a fluid containing various sized droplets.
In an example, a fluid containing oil, water and a surfactant is mixed so as to create a bulk emulsion. The bulk emulsion is injected into beginning of a microfluidic lateral diffusion device and various fractions are collected at the ending of the device at positions corresponding to specific sizes. Advantages to this lateral diffusion separation means would be the isolation of similarly-sized droplets off-line in a fast and facile manner. Bulk emulsions could be size-selected and then the resulting emulsions, if desired, combined to create sized libraries for re-introduction into a microfluidic device. In a further example, the lateral diffusion microfluidic devices could be rolled-up into a syringe or designed for parallel processing.
Recently, devices that exploit both techniques have been miniaturized with the use of microfabrication technology. Microfabricated devices have also been designed that inherently rely on diffusion for separation. Particle mixtures are either repeatedly subject to spatially asymmetric potentials created by microelectrodes or driven through arrays of micrometer-scale asymmetric obstacles to exploit differences in diffusion lengths. In all of the devices discussed so far, particles in a given zone have many different migration paths, and diffusion is required for separation.
The present invention describes a separation process that creates equivalent migration paths for each particle in a mixture, thereby eliminating multipath zone broadening (FIG. 24 ). FIG. 24 . (Panel A) Geometric parameters defining the obstacle matrix. A fluid flow is applied in the vertical direction (orange arrow). (Panel B) Three fluid streams (red, yellow, and blue) in a gap do not mix as they flow through the matrix. Lane 1 at the first obstacle row becomes lane 3 at the second row, lane 3 becomes lane 2 at the third row, and so on. Small particles following streamlines will thus stay in the same lane. (Panel C) A particle with a radius that is larger than lane 1 follows a streamline passing through the particle's center (black dot), moving toward lane 1. The particle is physically displaced as it enters the next gap. Black dotted lines mark the lanes.
The separation process uses laminar flow through a periodic array of micrometer-scale obstacles. Each row of obstacles is shifted horizontally with respect to the previous row by δλ, where λ is the center-to-center distance between the obstacles (FIG. 24 ). For convenience, let δλ/λ be ⅓. Fluid emerging from a gap between two obstacles will encounter an obstacle in the next row and will bifurcate as it moves around the obstacle. Let the flow diverted to the left of the obstacle be δΦ, where Φ is the total fluid flux going through the gap. If the fluid is confined to move straight down through the array, δ must equal δλ/λ. Let us then consider the flow through a gap to be made up of three lanes, each of which by definition has a flux of Φ/3. Because the Reynolds number is low (<103 in micrometer-scale environments) and flows are laminar, the streams in each lane do not cross or mix (FIG. 24B). Notably, as the lanes go through gaps, their positions relative to the gaps change. The lanes are represented in each gap by 1, 2, and 3, from left to right, respectively. Lane 1 becomes lane 3 in the next gap, lane 2 becomes lane 1, and lane 3 becomes lane 2 (FIG. 24 ). After three rows, the three lanes rejoin in their original configuration.
Particles that are smaller than the lane width will follow the streamlines. A particle starting in lane 1 will go through lane 3 (right lane with respect to the gap) in the second row, lane 2 (middle lane) in the third row, and back to lane 1 (left lane) in the fourth row (FIG. 24B). In fact, particles starting from any of the three lanes will go back to the original lane assignment after three rows, so that net migration is in the average flow direction. This motion is called the “zigzag mode.” In practice, particles can diffuse into an adjacent lane. However, the microscopic path for all lanes is equivalent, unlike the multiple paths particles take when moving through a column of porous beads. In contrast to the smaller particles, a particle with a radius larger than the width of lane 1 at a gap will behave differently in the array. This is because the center of the particle cannot “fit” into lane 1 in a gap. As such a particle from lane 2 in one gap moves into the subsequent gap, expecting to move through the gap in lane 1, the particle will be “bumped” and its center will thus be displaced into lane 2 (FIG. 24C). The particle will then flow with the fluid in lane 2. This process is repeated every time a large particle approaches a row of obstacles, so that the particle remains in lane 2 as it moves down through the array. This transport pattern is called the “displacement mode.” This is also applicable to electrophoresis by considering ion flows instead of fluid flows.
FIG. 25 shows High-resolution separation of fluorescent microspheres with diameters of 0.80 um (green), 0.90 um (red), and 1.03 um (yellow), with a matrix of varying gap size. Whereas the shift in registry and the lattice constants of the matrix remain the same, the obstacle diameters are changed to create gaps, d, of different sizes, which are labeled on the left side of the fluorescent image. The red bars on the fluorescence profile represent the width of the peaks (SD), and the black bars label the 1% inhomogeneity in the bead population. a.u., arbitrary units.
FIG. 26 is a schematic illustrating the separation by deterministic lateral displacement in an array of microposts, with an example row shift fraction of one-third. This shift creates three equal flux streamlines. The dashed lines are the boundaries between the streamlines, which are assigned an index in the gaps between the posts. Paths of particles both smaller and larger than the critical threshold are depicted with green and red dotted lines respectively. Small particles stay within a flow Stream and large particles are displaced at each obstacle. G is the clear spacing between the gap, is the center-to-center post separation, and d is the relative shift of the post centers in adjacent rows.
These described methods allow for the quick and efficient formation of uniformed sized droplet emulsion libraries for further use on a microfluidic device of the present invention.
Example 2
The present invention provides methods for performing polymerase chain reaction (PCR). PCR can be performed on a drop-by-drop basis in a microfluidic device according to the present invention. A monolithic chip can be provided wherein the heating and cooling lines are built into the chip and a sorting means is provided. Advantages of performing PCR in droplets on such a chip are that the chip is disposable and the reaction can be repeated without cleaning the device between reactions. Furthermore, the chip provides a convenient way of getting all the components to perform PCR in the droplets in the right concentration. Additionally, the PCR is more efficient because the heat transfer is more efficient due to the small volume. This provides for shorter incubation/residence 15 times. Droplets containing the nucleic acids, all PCR primers, and, if present, beads are generated one at a time at rates between 100 and 20,000 droplets per second. The droplets can then be sent through a serpentine path between heating and cooling lines to amplify the genetic material inside the droplets. Upon exiting the device the droplets may be sent for further on-chip or off-chip processing, directed into another chip, or the emulsion may be broken to release the PCR product. If present, beads may be harvested by passing the emulsion through a filtration device, sedimentation, or centrifugation.
The width and depth of the channel can be adjusted to set the residence time at each temperature, which can be controlled to anywhere between less than a second and minutes. At a typical rate of 1000 drops per second, 1 million strands of DNA can be amplified in approximately 20 minutes on one device. A typical flow rate of 250 μL/hour would correspond to 1000 drops of 50 microns in diameter being generated every second. Flow rates and droplet sizes can be adjusted as needed by controlling the nozzle geometry.
The present invention also provides methods for performing dideoxynucleotide sequencing reactions on a microfluidic device. Chain terminator sequencing (Sanger sequencing) is well known to those of ordinary skill in the art. DNA template preparation, cycling sequencing and preparing extension products for electrophoresis are related techniques and also well known to those of skill in the art. Applied Biosystems' “Automated DNA Sequencing: Chemistry Guide” 2000 is an excellent resource covering these techniques and is incorporated herein by reference in its entirety.
One method is to sequencing PCR templates which can include single amplification PCR or nested and semi-nested PCR strategies. In the simplest PCR sequencing case, the target DNA is amplified with a single set of primers and then sequenced using the same primers. For many samples, this works well. For the samples that do not work well with this method, optimization of the PCR amplification may be required. Optimizing the PCR minimizes the presence of non-specific product bands and ensures adequate yield. A single PCR amplification is also compatible with the use of a sequencing primer that binds internally (semi-nested or nested) to one or both of the PCR primers. This can be helpful if primer-dimer (primer oligomerization) artifacts are a problem.
If difficulty with more complex samples, such as bacterial genomic DNA, is encountered a nested or semi-nested PCR can be used. These techniques are useful when the target is present in small quantity. They offer more specificity, which provides superior sequencing data with reduced background signal. Both nested and semi-nested PCR require two amplifications. The first amplification is identical for nested and semi-nested, but the second amplification differs as described below. Amplify with one set of PCR primers, which converts a complex sample (such as bacterial genomic DNA) into a non-complex sample consisting of the first PCR product and some side products. Nested PCR: Amplify 1% or less of the first PCR reaction product using a second set of PCR primers that hybridize at positions internal to the first set. Semi-nested PCR: Only one primer of the second set of PCR primers is internal. The other primer is one of the original PCR primers.
A PCR primer can be synthesized with a universal sequencing primer binding site added to the 5′ end (e.g., see Appendix E in Applied Biosystems' “Automated DNA Sequencing: Chemistry Guide” for universal primer sequences). This allows any PCR product to be sequenced with universal primers. Universal-tailed PCR primers enable the use of commercially available dye-labeled sequencing primers. This technique is also useful with dye terminator chemistries, because universal sequencing primers have good annealing characteristics. However, the longer PCR primers add to the overall cost of the reactions. Using universal-tailed primers sometimes results in primer oligomerization. As these products have priming sites present, they can result in noisy data for the first 20-100 bases. Redesigning the PCR primer, optimizing the PCR amplification further, and employing Hot Start methods can help overcome this situation.
After PCR amplification, the resulting PCR product is in solution along with PCR primers, dNTPs, enzyme, and buffer components. The method used to prepare the PCR product for sequencing depends on the amounts of these components that are carried over and on the chemistry used for sequencing. Excess PCR primers carried over from the amplification reaction compete with the sequencing primer for binding sites and reagents in the sequencing reaction. This carryover of PCR primers presents more of a problem in dye terminator chemistries because the dye label is incorporated into the extension product after the primer anneals to the template. If more than one primer is present, multiple dye-labeled sequence ladders are generated, resulting in noisy data. Excess dNTPs from the amplification reaction can affect the balance of the sequencing reaction, resulting in decreased termination in shorter extension fragments.
Nonspecific PCR products include primer-dimer artifacts and secondary PCR products. The presence of any significant quantity of either in a PCR product can result in poor quality sequencing data. Nonspecific PCR products behave as templates in the sequencing reaction and produce extension products, which results in noisy data. These products often can be visualized on an agarose gel before sequencing. If they are present, the PCR amplification should be optimized and repeated before sequencing. Use of a nested or semi-nested sequencing primer can also allow good sequence data to be obtained. Alternatively, the PCR product of interest can be purified by agarose gel electrophoresis.
There are several ways to minimize contaminants in a PCR amplification: PCR optimization (Innis and Gelfand, 1990): (1) Amount of starting DNA; (2) Careful primer design; (3) Primer concentration, (4) Enzyme concentration, (5) Magnesium ion (Mg2+) concentration, (6) Nucleotide concentration; (7) Buffer composition; (8) Number of cycles; (9) pH; (10) Manual Hot Start method; (11) AmpliTaq Gold® DNA Polymerase as an automatic Hot Start and/or (12) Limiting dNTPs and primers. All of these methods increase the specificity of the PCR amplification and decrease the amount of contaminants that can interfere with a sequencing reaction.
There are several methods for purifying PCR products: (1) Column purification; (2) Ethanol precipitation; and/or (3) Gel purification.
An alternative to one of the more stringent purification methods listed above is treatment of PCR products with shrimp alkaline phosphatase (SAP) and exonuclease I (Exo I) before sequencing. The SAP/Exo I procedure degrades nucleotides and single-stranded DNA (primers) remaining after PCR (Werle et al., 1994). This procedure is particularly useful in cases where limiting concentrations of primers and nucleotides cannot be used for direct PCR sequencing.
FIG. 27 shows one embodiment for a DNA sequencing chip design. Template DNA and primers are combined at step ‘add 1’ and the reaction is incubated at 95° C. for a hot start (position 1). The reaction then cycles 20-30 times (position 2) before the addition of SAP and ExoI at ‘add 2.’ The reaction is incubated at 37° C. for a pre-defined time-period and then the SAP and Exol enzymes are inactivated at 95° C. (position ‘4’). The SAP/ExoI procedure degrades nucleotides and single-stranded DNA (primers) remaining after PCR. The universal sequencing primers, ddNTPs and buffers are added at ‘add 3,’ and the PCR sequencing reaction is allowed to proceed at position ‘5.’ The final reaction product is collected and can be stored off-chip.
Step Action
1. For each sample, combine the following:
    • SAP (1 Unit/μL), 2 μL
    • Exo I (10 Units/μL), 0.2 μL
    • Deionized water, 6.0 μL
    • Note In general this procedure works well using 0.5 units of each enzyme per microliter of PCR products used. The procedure seems to work equally well with or without the use of SAP buffer, so this has been excluded in this protocol.
2. Add 4.0 μL of PCR product to the above mix.
3. Incubate at 37° C. for 1 hour.
4. Incubate at 72° C. for 15 minutes to inactivate the enzymes.
The recommended DNA quantities for sequencing reactions are shown in Table 3-1 below.
TABLE 3-1
Recommended Ranges of DNA Template Quantity for Each Chemistry
Cycle Sequencing Chemistry
Rhodamine Fluorescein/
Dye dRhodamine BigDye Rhodamine BigDye
Template Terminator Terminator Terminator Dye Primer Primer
PCR product:
100-200 bp   1-3 ng   1.-3 ng   1-3 ng   2-5 ng   2-5 ng
200-500 bp   3-10 ng  3-10 ng   3-10 ng   5-10 ng   5-10 ng
600-1000 bp    5-20 ng  5-20 ng   5-20 ng  10-20 ng  10-20 ng
1000-2000 bp   10-40 ng  10-40 ng  10-40 ng  20-50 ng  20-50 ng
  >2000 bp  40-100 ng 40-100 ng  40-100 ng  50-150 ng  50-150 ng
single- 100-250 ng 50-100 ng  50-100 ng 150-300 ng 150-400 ng
stranded
double- 200-500 ng 200-500 ng  200-500 ng 300-600 ng 200-800 ng
stranded
coamid, BAC  0.5-2.0 μg not  0.6-1.0 μg  0.5-2.0 μg  0.5-1.0 μg
recommended
bacterial not recommended   2-3 μg not recommended
genomic DNA
PCR protocols that limit amounts of primers and dNTPs allow the product of the reaction to be used for sequencing with no purification. This is usually carried out by setting up the PCR amplification with 5-10 pmol of primers and 20-40 μM dNTPs, so that most of the primers and dNTPs are exhausted during amplification. If the yield of the desired PCR product is high and the product is specific, i.e., it produces a single band when analyzed by agarose gel electrophoresis, the sample can be diluted before sequencing and will give good results. The dilution ratio depends on the concentration of your PCR product and needs to be determined empirically (start with 1:2 and 1:10 dilutions with deionized water). When you limit concentrations of primers and dNTPs and dilute the PCR products, the PCR parameters have to be robust. Direct PCR sequencing is most useful in applications where the same target is being amplified and sequenced repeatedly and PCR conditions have been optimized. Direct PCR sequencing can be done with dye primer chemistries. With dye terminator chemistries, it is much more critical that the PCR primers be consumed. Excess PCR primers will be extended and labeled by the cycle sequencing reaction, resulting in noisy data. Direct PCR sequencing does not work for XL PCR because limiting amounts of primers and dNTPs cannot be used. The PCR product should be purified or the excess primers and nucleotides should be degraded by SAP/Exo I treatment.
Example 3
The present invention provides methods for performing isothermal-type amplification methods on a microfluidic device. Isothermal amplification is an alternative to the standard PCR techniques described herein. Isothermal amplification is used to reduce the relative amount of background DNA in a sample. Primers are generally used in a constant temperature means of amplification. Isothermal amplification is applicable for SNP detection. Once the DNA is amplified by isothermal amplification there are several well-known means for detecting which nucleotide polymorphism is present. These include, but are not limited to; allele specific primer extension, oligonucleotide ligation assay, mini-sequencing, fluorescence polarization, etc. Isothermal amplification is also applicable for DNA sequencing preparation. The isothermally-amplified DNA can be attached to a solid phase within a droplet or placed within a parking space on chip. The beads or parking spaces can be accessed and the amplified DNA used for a DNA sequencing reaction. Further, isothermal amplification is applicable for gene expression analysis. Isothermal amplification can be used to monitor gene expression by the measurement of the amount of cDNA produced in a quantitative fashion. Many methods for isothermal amplification are known in the art, including but not limited to the following examples.
Rolling circle amplification (RCA). A DNA polymerase extends a primer on a circular template, generating tandemly linked copies of the complementary sequence of the template (Fire & Xu, 1995). The TempliPhi amplification process using rolling circle amplification is known in the art. In the process, random hexamer primers anneal to the circular template DNA at multiple sites. Phi29 DNA polymerase extends each of these primers. When the DNA polymerase reaches a downstream-extended primer, strand displacement synthesis occurs. The displaced strand is rendered single-stranded and available to be primed by more hexamer primer. The process continues, resulting in exponential, isothermal amplification.
Transcription mediated amplification (TMA). An RNA polymerase is used to make RNA from a promoter engineered in the primer region, a reverse transcriptase to produce complementary DNA from the RNA templates and RNase H to remove the RNA from cDNA (Guatelli et al, 1990).
Strand-displacement amplification (SDA). A restriction endonuclease is used to nick the unmodified strand of its target DNA and the action of an exonuclease-deficient DNA polymerase to extend the 30 end at the nick and displace the downstream DNA strand (Walker et al, 1992). Strand-displacement amplification is known in the art.
Helicase-dependent amplification (HDA). A DNA helicase is used to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. Schematic diagram of HAD is shown in FIG. 28 . Two complementary DNA strands are shown as two lines: the thick one is the top strand and the thin one is the bottom strand. 1: A helicase (black 30 triangle) separates the two complementary DNA strands, which are bound by SSB (grey circles). 2: Primers (lines with arrow heads) hybridize to the target region on the ssDNA template. 3: A DNA polymerase (squares with mosaic patterns) extends the primers hybridized on the template DNA. 4: Amplified products enter the next round of amplification.
One example is emulsion-based sample preparation. Random libraries of DNA fragments are generated by shearing an entire genome and isolating single DNA molecules by limiting dilution. See, FIG. 29 . Specifically, sequencing reactions such as those performed by Solexa, 454 Life Sciences and others involve randomly fragmenting the entire genome, adding specialized common adapters to the fragments, capturing the individual fragments on their own beads and, within the droplets of an emulsion, clonally amplifying the individual fragment (FIG. 29 a, 29 b ). Unlike in current sequencing technology, their approach does not require subcloning or the handling of individual clones; the templates are handled in bulk within the emulsions. Typically, about 30% of the beads will have DNA, producing 450,000 template-carrying beads per emulsion reaction.
Sample preparation and DNA sequencing is shown in FIG. 29 . Panel A, Genomic DNA is isolated, fragmented, ligated to adapters and separated into single strands (top left). Fragments are bound to beads under conditions that favor one fragment per bead, the beads are captured in the droplets of a PCR-reaction-mixture-in-oil emulsion and PCR amplification occurs within each droplet, resulting in beads each carrying ten million copies of a unique DNA template (top, second from the left). The emulsion is broken, the DNA strands are denatured, and beads carrying single-stranded DNA clones are deposited into wells of a fiber-optic slide (bottom left). Smaller beads carrying immobilized enzymes required for pyrophosphate sequencing are deposited into each well (bottom, second from the left). Panel B, Microscope photograph of emulsion showing droplets containing a bead and empty droplets. The thin arrow points to a 28-mm bead; the thick arrow points to an approximately 100-mm droplet. Panel C, Scanning electron micrograph of a portion of a fiber-optic slide, showing fiber-optic cladding and wells before bead deposition. Panel D, The sequencing instrument consists of the following major subsystems: a fluidic assembly. Panel E, a flow chamber that includes the well-containing fiber-optic slide. Panel F, a CCD camera-based imaging assembly. Panel G, and a computer that provides the necessary user interface and instrument control.
Another example is sequencing in fabricated picolitre-sized reaction vessels. One method uses sequencing by synthesis simultaneously in open wells of a fiber-optic slide using a modified pyrosequencing protocol that is designed to take advantage of the small scale of the wells. The fiber optic slides are manufactured by slicing of a fiber-optic block that is obtained by repeated drawing and fusing of optic fibers. At each iteration, the diameters of the individual fibers decrease as they are hexagonally packed into bundles of increasing cross-sectional sizes. Each fiber-optic core is 44 μm in diameter and surrounded by 2-3 μm of cladding; etching of each core creates reaction wells approximately 55 μm in depth with a centre-to-centre distance of 50 μm (FIG. 29 c ), resulting in a calculated well size of 75 pl and a well density of 480 wells per square mm. The slide, containing approximately 1.6 million wells, is loaded with beads and mounted in a flow chamber designed to create a 300-μm high channel, above the well openings, through which the sequencing reagents flow (FIG. 29 d ). The unetched base of the slide is in optical contact with a second fiber optic imaging bundle bonded to a charge-coupled device (CCD) sensor, allowing the capture of emitted photons from the bottom of each individual well (FIG. 29 d ). A three-bead system has been developed and the components optimized to achieve high efficiency on solid support. The combination of picoliter-sized wells, enzyme loading uniformity allowed by the small beads and enhanced solid support chemistry enabled users to develop a method that extends the useful read length of sequencing-by-synthesis to 100 bases.
In the flow chamber cyclically delivered reagents flow perpendicularly to the wells. This configuration allows simultaneous extension reactions on template-carrying beads within the open wells and relies on convective and diffusive transport to control the addition or removal of reagents and by-products. The timescale for diffusion into and out of the wells is on the order of s in the current configuration and is dependent on well depth and flow channel height. The timescales for the signal-generating enzymatic reactions are on the order of 0.02-1.5 s. The current reaction is dominated by mass transport effects, and improvements based on faster delivery of reagents are possible. Well depth was selected on the basis of a number of competing requirements: (1) wells need to be deep enough for the DNA-carrying beads to remain in the wells in the presence of convective transport past the wells; (2) they must be sufficiently deep to provide adequate isolation against diffusion of by-products from a well in which incorporation is taking place to a well where no incorporation is occurring; and (3) they must be shallow enough to allow rapid diffusion of nucleotides into the wells and rapid washing out of remaining nucleotides at the end of each flow cycle to enable high sequencing throughput and reduced reagent use. After the flow of each nucleotide, a wash containing a pyrase is used to ensure that nucleotides do not remain in any well before the next nucleotide being introduced.
Another example is base calling of individual reads. Nucleotide incorporation is detected by the associated release of inorganic pyrophosphate and the generation of photons. Wells containing template-carrying beads are identified by detecting a known four-nucleotide ‘key’ sequence at the beginning of the read. Raw signals are background-subtracted, normalized and corrected. The normalized signal intensity at each nucleotide flow, for a particular well, indicates the number of nucleotides, if any, that were incorporated. This linearity in signal is preserved to at least homopolymers of length eight. In sequencing by synthesis a very small number of templates on each bead lose synchronism (that is, either get ahead of, or fall behind, all other templates in sequence). The effect is primarily due to leftover nucleotides in a well (creating ‘carry forward’) or to incomplete extension. Typically, a carry forward rate of 1-2% and an incomplete extension rate of 0.1-0.3% is seen. Correction of these shifts is essential because the loss of synchronism is a cumulative effect that degrades the quality of sequencing at longer read lengths.
Methods have demonstrated the simultaneous acquisition of hundreds of thousands of sequence reads, 80-120 bases long, at 96% average accuracy in a single run of the instrument using a newly developed in vitro sample preparation methodology and sequencing technology. With Phred 20 as a cutoff, they are able to show that their instrument is able to produce over 47 million bases from test fragments and 25 million bases from genomic libraries. Recent work on the sequencing chemistry and algorithms that correct for crosstalk between wells suggests that the signal distributions will narrow, with an attendant reduction in errors and increase in read lengths. In preliminary experiments with genomic libraries that also include improvements in the emulsion protocol, one is able to achieve, using 84 cycles, read lengths of 200 bases with accuracies similar to those demonstrated here for 100 bases. On occasion, at 168 cycles, individual reads that are 100% accurate over greater then 400 bases have been generated.
Isothermal amplification reactions, as described above, have shown great promise generating high yields with great fidelity. However, an associated drawback arises from the tendency of the polymerase to generate spurious, non-template amplification products when reactions are conducted in the absence of template DNA. Additionally, our application utilizes high microfluidic throughput in conjunction with limiting DNA template dilutions to amplify single template molecules. As a result, 10 the number of empty reaction droplets increases considerably, comprising 90% or more of the total droplet population following Poisson distributions. Non-template amplification (hereafter NTA) in even a small fraction of the total droplets can confound amplification detection strategies based on laser interrogation of intercalating dyes, thus this issue must be resolved. To address this problem in the art, the present invention provides the use of mixed modified and standard hexamer primers in microfluidic reactions to retard NTA while allowing template-based amplification to proceed.
Previous work has attempted to reduce NTA through incorporation of nitroindole bases (Loakes and Brown 1994; Loakes, Hill et al. 1997) in the random primers (Lage, Leamon et al. 2003) or reducing reaction volumes to 600 nL (Hutchison, Smith et al. 2005). Unfortunately, modified nitroindole primers have proven difficult to replicate, and often have the effect of significantly reducing the overall rate and yield of the amplifications in which they are incorporated. Low volume reactions conducted in multiplate wells have encountered difficulties stemming from dispensation of low volumes, and associated issues of sample evaporation, well to well contamination, etc.
The modified primers of the present invention containing nitroindoles and C3 non-replicable elements were studied in an effort to reduce NTA both in bulk and microfluidic reactions. Both nitroindoles and C3 non-replicable elements were found to be effective in reducing NTA, with primers containing two 5′ nitroindoles most effective in NTA suppression. However, increased NTA suppression was tightly linked with reduced yield in template amplification reactions. Amplifications using a ratio of nitroindole to random hexamer primers generated a range of both template and non-template amplification yields, with a 15:85 ratio of nitroindole to random hexamers generating template yields commensurate with random hexamers primers alone, but generating little if any spurious product in the absence of template.
Example 4
The PCR and isothermal amplifications described herein can be very useful in performing single nucleotide polymorphism analysis. A Single Nucleotide Polymorphism, or SNP, is a small genetic change, or variation, that can occur within a person's DNA sequence. The genetic code is specified by the four nucleotide “letters” A (adenine), C (cytosine), T (thymine), and G (guanine). SNP variation occurs when a single nucleotide, such as an A, replaces one of the other three nucleotide letters—C, G, or T.
An example of a SNP is the alteration of the DNA segment AAGGTTA to ATGGTTA, where the second “A” in the first snippet is replaced with a “T”. On average, SNPs occur in the human population more than 0.1 percent of the time. Because only about 3 to 5 percent of a person's DNA sequence codes for the production of proteins, most SNPs are found outside of “coding sequences”. SNPs found within a coding sequence are of particular interest to researchers because they are more likely to alter the biological function of a protein. Because of the recent advances in technology, coupled with the unique ability of these genetic variations to facilitate gene identification, there has been a recent flurry of SNP discovery and detection.
Although many SNPs do not produce physical changes in people, scientists believe that other SNPs may predispose people to disease and even influence their response to drug regimens. Currently, there is no simple way to determine how a patient will respond to a particular medication. A treatment proven effective in one patient may be ineffective in others. Worse yet, some patients may experience an adverse immunologic reaction to a particular drug. Today, pharmaceutical companies are limited to developing agents to which the “average” patient will respond. As a result, many drugs that might benefit a small number of patients never make it to market.
In the future, the most appropriate drug for an individual could be determined in advance of treatment by analyzing a patient's SNP profile. The ability to target a drug to those individuals most likely to benefit, referred to as “personalized medicine”, would allow pharmaceutical companies to bring many more drugs to market and allow doctors to prescribe individualized therapies specific to a patient's needs.
Finding single nucleotide changes in the human genome seems like a daunting prospect, but over the last 20 years, biomedical researchers have developed a number of techniques that make it 25 possible to do just that. Each technique uses a different method to compare selected regions of a DNA sequence obtained from multiple individuals who share a common trait. In each test, the result shows a physical difference in the DNA samples only when a SNP is detected in one individual and not in the other. Currently, existing SNPs are most easily studied using microarrays. Microarrays allow the simultaneous testing of up to hundreds of thousands of separate SNPs and are quickly screened by computer.
The race among pharmaceutical companies today is to apply new system genomics approach to identify novel targets and validate these targets in the most efficient fashion. SNP research will provide fundamental understanding of many polygenic diseases, thus providing new therapeutic targets. As groups have performed genome-wide scans and other large studies that require the genotyping of thousands of SNPs and samples, a need for high-throughput SNP genotyping has become essential.
The microfluidic device of the present invention is capable of performing at least 10,000 SNP analysis per second, such that a full genome scan (i.e., 100K SNPs) with 10× overrepresentation can be performed in less than an hour. The speed and efficiency permitted using the devices and methods of the present invention will significantly lower the associated costs and reagent consumption for performing SNP analysis.
Example 5
The PCR and isothermal amplifications described herein can be very useful in providing necessary sample preparation processes for commercial available DNA sequencers, such as the Solexa's 1G sequencer, which relies upon immobilized DNA for a substrate. In one embodiment, single molecules can be amplified using PCR primers treated with moieties used for surface immobilization, then flow the PCR positive droplets across the surface of the slide, forming a packed emulsion. The emulsion can then be broken, and the primers allowed to bind to the slide due the presence of the appropriate coating to bind the PCR products.
The Solexa 1G sequencer currently sequences amplified material that has been amplified in place, on primers bound to the slide, by bridge amplification. In this process, template DNA is flowed across the slide surface at very low concentrations, and adapters previously ligated to each template hybridize to complimentary primers attached to the slide in any one of eight lanes. Once hybridized, the primers are subjected to bridge amplification, a version of PCR amplification that utilizes immobilized primers—the product of the reaction is a patch of DNA immobilized to the slide. Several risks are encountered when amplifying DNA in this matter—the process is exquisitely sensitive to DNA concentration—if too much DNA is used, the DNA patches will be generated too closely, or even overlap, generating mixed signals during subsequent sequencing. If too little DNA is used, the DNA patches will be present at a very low density, and insufficient sequence may be generated during the run. As the sequencing reactions take 72 hours, and no titration runs are conducted to test the DNA concentrations prior to amplification, the potential loss of time and money is considerable. Additionally, neither single molecule amplification nor bridge amplification are very efficient, and bridge amplification has an upper and lower size limitation, generating products only within a particular length range.
The present invention provides methods to overcome this limitations. In one embodiment, a Solexa slide can be coated with any of the compounds commonly used to permit binding and immobilization (e.g., carboxy-esters, streptavidin, Igg, gold, etc.). PCR reactions could be performed as described in the instant microfluidic device using primers modified with the appropriate binding moiety (5′ amines, 5′ biotins, 5′ DNPs or 5′ Thiols, respectively) to efficiently amplify PCR products in solution which could then be efficiently and easily bound to the Solexa slide for subsequent sequencing. The amplification is quite straight forward, conducted with a limiting dilution of template and a set of primer pairs compatible with the adapters ligated to the Solexa templates through their standard sample preparation process. One of the primer pairs would possess a 5′ binding moiety as described earlier, only one of the pair as this will permit removal of the opposing strand and the generation of single stranded immobilized templates on the slide. Once amplification has been conducted, and the positive droplets sorted, the droplets can be flowed onto each of the lanes of the Solexa slide. Proper spacing between the droplets can be obtained by mixing droplets containing only buffer or immiscible oil in with the PCR positive droplets at a ratio sufficient to ensure that the PCR positive droplets are rarely proximal to each other. Once each lane has been packed with droplets, the droplets can be broken through the application of electrical field and the PCR products allowed to bind to the slide in the same geographic area that the droplet had occupied. The immobilized templates can be rendered single stranded through the application of basic washes, temperature etc. This will permit the rapid amplification of PCR fragments and their subsequent density-controlled deposition onto the Solexa chip for sequencing.
Example 6
The present invention provides methods for detecting the presence and/or sequence of nucleic acids in low copy number in droplets on a microfluidic device. The detection of a specific nucleic acid sequence present in a sample by probing the sample with a complementary sequence of nucleic acids is a well known technique. Nucleic acids are highly specific in binding to complementary nucleic acids and are thus useful to determine whether a specific nucleic acid is present in a sample. One must know the sequence of the specific nucleic acid to be detected and then construct a probe having a complementary nucleic acid sequence to the specific nucleic acid sequence.
Since nucleic acid probes are highly specific, it is preferable in some situations to probe the nucleic acid sequence itself rather than the protein produced by the nucleic acid sequence. As a particular example, a diagnostic method based solely on protein detection would be unreliable for determining the presence of infectious particles of hepatitis B virus, due to the presence of significant levels of non-infectious antigen particles which lack the DNA genome. In another example, the various subtypes of human papilloma virus found in either pre-cancerous or benign cervical tumors can be distinguished only by the use of nucleic acid probe hybridization. Also, the specific genetic makeup of an AIDS virus makes it certain that an assay based on the presence of an AIDS virus specific nucleic acid sequence would be superior as a diagnostic.
The naturally-occurring high number of ribosomal RNA, up to 100,000 copies per cell, has been used by GenProbe to facilitate diagnosis of certain bacterial pathogens, such as Legionella and Mycoplasma, using nucleic acid probes. However, this strategy cannot be used with non-cellular pathogens, such as viruses, or with probed nucleic acid sequences with low copy numbers. Copy number is a particular problem with the development of a nucleic acid probe method for the detection of AIDS virus, where the integrated provirus may be present in less than one of ten thousand peripheral blood lymphocytes. Thus, if the particular nucleic acid sequence suspected to be present in a sample could be amplified, the copy number problem could be circumvented and probe assays could be more readily used.
In a normal biological sample, containing only a few cells, and consequently only a few copies of a particular gene, it is necessary to utilize an amplification process in order to overcome the 5 copy number problem.
One method to amplify is to ‘grow out’ the sample, that is, to arrange conditions so that the living biological material present in the sample can replicate itself. Replication could increase the quantity of nucleic acid sequences to detectable levels. In the food industry, for example, in order to test processed food for the food-poisoning bacteria Salmonella, food samples must be incubated for a number of days to increase the quantity of nucleic acid copy numbers. In clinical samples, pathogens must also be allowed to increase their number by growing out over some considerable time.
Current methods utilize a process in which a sample suspected of containing a target DNA sequence is treated with oligonucleotide primers such that a primer extension product is synthesized which in turn serves as a template, resulting in amplification of the target a DNA sequence. The primer extension product is separated from the template using heat denaturation. Current methods also include a process for amplifying a target DNA sequence having two separate complementary strands. The process includes treating the strands with primers to synthesize extension products, separating the primer extension products from the templates, and in turn using the primer extension products as templates.
Both of the above methods require either manual or mechanical participation and multi-step operations by the user in the amplification process and are restricted to amplifying DNA only. The steps involved in these methods require the user to heat the sample, cool the sample, add appropriate enzymes and then repeat the steps. The temperature changes cause the enzymes to loose their activity. Hence, the user is required to repeatedly supplement the amplification mixture with aliquots of appropriate enzymes during the amplification process.
The present invention provides methods for detecting the presence and/or sequence of nucleic acids in low copy number in droplets on a microfluidic device. In one embodiment of the invention, a nucleotide/peptide nucleic acid (pna) probe (oligo probe) can be tethered such that when it binds to a template DNA molecule in low copy number located inside an aqueous emulsion (i.e., droplet) it turns on or activates an enzyme. By way of nonlimiting example, an alkaline phosphatase conjugate can be placed on one and of the low copy number nucleotide, and an inhibitor to alkaline phosphatase on the other end (like a molecular beacon). When the oligo probe binds to the low copy number template the inhibitor is removed from the enzyme and the enzyme turns over the substrate. The tethers can be a protein complementation assay wherein the binding of the oligo probe to the low copy number template causes the enzyme to be active.
Various other embodiments are described herein and should not be considered as limiting to the invention. Example 1: Taqman chews a beta-gal alpha protein attached at the 3′ end of an oligo probe thereby releasing free alpha subunit to bind to omega fragment in solution. Example 2: Two oligo probes sit down on the low copy number template adjacent to each other, thereby bringing two subunits of a protein complementing assay reagent together. Example 3: A Taqman-like enzyme that releases an active moiety can also be used. The active moiety can include, for example, an enzyme that becomes activated upon release from the oligo probe, or a fluorescent group that is quenched while tethered to the oligo probe. The use of a double-strand specific nuclease that will chew up the probe only when the probe is bound, thereby releasing active enzyme or fluorescent substrates. Example 4: The probe has a fluorescent group attached such that the detected hybridization causes the release of the fluorescent group a la Taqman. Example 5: The probe has an inactive enzyme attached such that the detected hybridization causes the release of the active enzyme by a Taqman-type release. Example 6: The probe has an inactive complementing enzyme attached such that the detected hybridization causes the release of the active moiety of the enzyme to be able to complement. Example 7: Two probes have inactive enzyme moieties attached such that the detected hybridization causes the complementation and activation of the enzyme. Example 8: Two probes come together and allow a Fluorescence Resonance Energy Transfer (FRET) reaction to occur. This would require a FRET-oligo library. Almost all SNP or transcriptional profiling method may be amenable to this concept.
Many assays are conducted on microfluidic devices are including, but not limited to, protein-protein, antibody-antigen, nucleic acid-protein, nucleic acid-nucleic acid, ligand-protein, ligand nucleic acid, ligand-ligand, eukaryotic or prokaryotic cell surface moiety-second moiety, the measurement of two or more receptors on the surface of an eukaryotic or prokaryotic cell, the development of three-hybrid type systems using tandem fusions, interactor-cofactor, etc. Many other types of interactions are also known that can be adapted to the system. Assays incorporating complementation assays can be used in both proteomics and genomics.
Currently, these assay methods require methods require >10K fluorescein molecules to detect interaction/binding. In an embodiment, the present invention provides methods which reduce the number of interacting proteins by amplifying the signal of molecules that do interact. In preferred embodiments of some assays, stable emulsions may not be needed. An advantage is that we can eliminate surfactants and stabilizing additives which can affect protein activity within droplets.
Likewise, one could conceivably look for loss of complementation in any of the above assays. This loss or gain of complementation can be a function of physical (e.g., heat, light) or chemical additions to, or formulation of the droplets. These interactions include, but are not limited to, protein-protein, antibody-antigen, nucleic acid-protein, nucleic acid-nucleic acid, ligand-protein, ligand-nucleic acid, ligand-ligand, eukaryotic or prokaryotic cell surface moiety-second moiety, the measurement of two or more receptors on the surface of an eukaryotic or prokaryotic cell, the development of three-hybrid type systems using tandem fusions, interactor-cofactor, etc. Many other types of interactions are also known that can be adapted to the system. Assays incorporating complementation assays can be used in both proteomics and genomics.
The amount of each interacting partner may be with-in drop quantifiable by genetically or chemically coupling a reporter molecule (e.g., a dye or quantum dot, GFP protein) to one and or the other. Similarly, the complementation assay described for enzymatic amplification can use one of several different complementing proteins such that the concentration of each partner can be calculated within the droplet by using different enzyme substrates added to the droplets at the same or differing time. Timing of substrate addition is not critical and one of skill in the art would readily recognize that addition can be done at different times. ‘Killing’ by various denaturants, protease, etc. is also within the purview of the skilled artisan.
In an embodiment, complementation assays can be used to add a specific addition to, for example, an IVT synthesized protein. As an example, the s-peptide of RNaseA and the S protein wherein the S peptide is genetically fused to the IVT-generated Ab and the S protein, upon binding to the S peptide activates the RNaseA activity and thereby stops further IVT synthesis. Similarly, any two complementing interactrs can be used to generate an activity.
By quantifying the amount of interactors in a droplet it may be possible to derive interaction kinetics (affinity and disassociation constants as examples). In an embodiment, the present invention provides methods which can allow the quantification of proteins below that which can be seen by fluorescent spectroscopy in the absence of amplification. Alternatively, amplification may also not be 20 needed if one can genetically add a quantified number of reporters to the end of a molecule. For example, genetically fusing 10 GFP proteins onto the end of a protein would thereby increase the fluorescence intensity 10-fold. Similarly, a series of small complementing moieties can be fused onto the end of a protein and there obviate the need for long genetic fusions. As an example, a series of 10 s peptides spaced apart by a linker would be able to each ‘grab hold’ of an S protein to generate an increased signal over that which can be achieved by a single enzyme. Other examples include either biotin or biotin-binding protein mimetic and streptavidin or avidin, FLAG tags, poly histidines, complementing GFP, etc. Another example includes Qcoding the droplets.
FIGS. 30 and 31 show the current method for isolating antibodies on a microfluidic device. In the current method, DNA beads are made using bulk emulsion PCR; DNA-containing beads preferably isolated before loading onto a microfluidic device. The antigen can be <20 kD, (preferably <10 kD) and labeled with an appropriate dye, (preferably with several dye-molecules). It may even be preferable to put a poly-lysine tail onto the antigen to increase signal (but be concerned about quenching). The antigen is formulated into the bead-containing droplets at a concentration of 100 nm to allow proper sensitivity in the droplet (use of multiple/different dyes may allow this concentration to drop). In some situations it is possible to add antigen at the same time as the IVT solution.
FIG. 32 shows the method of the present invention for isolating antibodies on the microfluidic device. The right panel is a diagram of individual steps proposed to amplify signal of interacting antibody and antigen. The left panel is a schematic as would be designed for a chip to be used on microfluidic device.
FIG. 33 shows the genetic selection for full length antibody clones. A genetic selection can be used to enrich for full-length antibody clones by transforming E. coli and selecting for clones able 5 to grow on medium in which a suitable sugar is the only carbon source.
Example 7
The present invention provides a microfluidic device topology and implementation that merges the functionality of a first microfluidic substrate with that of a second microfluidic substrate by using forced withdrawal and reinjection into the same fluidic port. In current multi-step assays, the following steps must be performed to complete a “two step” experiment: (1) Installation of the first microfluidic substrate; (2) Installation of all first stage reagents; (3) Priming of all fluid lines and stabilization of the device operation; (4) Passive connection of a storage container to the instrument after device has been stabilized; (5) Disconnection of the storage container from the first device when collection complete; (6) Incubation of the collected emulsion; (7) Removal of the first device from the instrument; (8) Cleaning of the fluid lines needed for the second step of the experiment; (9) Installation of a second device to the instrument; (10) Connection of all second stage reagents, including the emulsion collected during the first stage; (11) Priming of the fluidic connections to the second device prior to running the second half of the measurement; and (12) Collection/readout of the second stage.
The methods of the present invention replaces current methodology of multi-step assays with the following: (1) Installation of the first microfluidic substrate; (2) Installation of all first and second stage reagents, including the first stage storage container (if it is not already connected); (3) Priming and stabilization of the fluidic lines and device; (4) Controlled collection of the first stage combined droplets by actively withdrawing some fraction of the oil and all of the generated emulsion into the storage container; (5) Incubation of the collected emulsion; (6) Startup and reinjection of the collected droplets back into the second half of the device; and (8) Collection/readout of the second stage. FIG. 34 depicts a schematic representation of this device topology.
Elimination of the handling of the collected emulsion has significant benefits beyond simplifying the user interaction with the instrument. Contaminants have the potential to ruin the experiment, and any extra handling and connection/disconnection increase the probability that contaminants will be introduced into the instrument.
Example 8
Gene silencing through RNAi (RNA-interference) by use of short interfering RNA (siRNA) has emerged as a powerful tool for molecular biology and holds the potential to be used for therapeutic gene silencing. Short hairpin RNA (shRNA) transcribed from small DNA plasmids within the target cell has also been shown to mediate stable gene silencing and achieve gene knockdown at levels comparable to those obtained by transfection with chemically synthesized siRNA (T. R. Brummelkamp, R. Bernards, R. Agami, Science 296, 550 (2002), P. J. Paddison, A. A. Caudiy, G. J. Hannon, PNAS 99, 1443 (2002)). Possible applications of RNAi for therapeutic purposes are extensive and include silencing and knockdown of disease genes such as oncogenes or viral genes.
Many assays are conducted on microfluidic devices are including, but not limited to, protein-protein, antibody-antigen, nucleic acid-protein, nucleic acid-nucleic acid, ligand-protein, ligand-nucleic acid, ligand-ligand, eukaryotic or prokaryotic cell surface moiety-second moiety, the measurement of two or more receptors on the surface of an eukaryotic or prokaryotic cell, the development of three-hybrid type systems using tandem fusions, interactor-cofactor, etc. Many other types of interactions are also known that can be adapted to the system. However, there is a need in the art for improved methods of RNAi screening, quickly and accurately.
The present invention provides methods for the screening of lethal and synthetic lethal RNAi-induced phenotype on a microfluidic device. The present invention utilizes a lentiviral library of RNAi where each virus has a unique 60-nt identifying barcode bracketed on either side with nucleotide sequences common to all vectors.
The analysis of lethal and synthetic lethal RNAi-induced phenotypes occurs in two steps. In the first step, the viral library is combined in bulk and infected, also in bulk, into an appropriate host strain. The molar amount of each of the different lentivirus in the library is pre-determined by sequencing on, for example, an appropriate instrument or by gene expression analysis on a microfluidic device. Post infection, the treated cells are collected and the 60-nt barcode is amplified from chromosomal DNA using PCR primers based on the bracketing sequence. In the second step, the PCR amplification product is added to a microfluidic device and analyzed against a labeled droplet library wherein the labeled droplets contain lentiviral-barcode-quantification reagents (e.g., molecular beacons, Taqman probes, etc.) against each of said lentiviral barcodes. A gene-expression analysis-like analysis is performed to quantify the amount of each lentiviral barcode-type in the treated cells. An absence or significant decrease of any lentiviral barcode in the amplified product can be assumed to be due to the death of that barcode-containing lentivirus in the treated cells. In an embodiment, the products within the droplets can also be amplified.
GFP or additional transcription analysis can also occur in two steps. In step one, the viral library is combined in bulk and infected, also in bulk, into an appropriate host strain. The molar amount of each of the different lentivirus in the library is pre-determined by sequencing on, for example, an appropriate instrument or by gene expression analysis on a microfluidic device. Post infection, the treated cells are i) collected in bulk, ii) sorted using a phenotype able to be sorted in a microfluidic device (e.g, GFP expression, cell-surface marker, low-copy cell-surface marker, etc.) and iii) the 60-nt barcode is amplified from chromosomal DNA using PCR primers based on the bracketing sequence. In the second step, the PCR amplification product is added to a microfluidic device and analyzed against a labeled droplet library wherein the labeled droplets contain lentiviral-barcode-quantification reagents (e.g., molecular beacons, Taqman probes, etc.) against each of said lentiviral barcodes. A gene-expression analysis-like analysis is performed to quantify the amount of each lentiviral barcode-type in the treated cells. An absence or significant decrease of any lentiviral barcode in the amplified product can be assumed to be due to the death of that barcode-containing lentivirus in the treated cells.
Example 9
Many diseases are associated with particular chromosomal abnormalities. For example, chromosomes in cancerous cells frequently exhibit aberrations called translocations, where a piece of one chromosome breaks off and attaches to the end of another chromosome. Identifying such chromosome abnormalities and determining their role in disease is an important step in developing new methods for diagnosing many genetic disorders. Traditional karyotyping using Giemsa staining allows scientists to view the full set of human chromosomes in black and white, a technique that is useful for observing the number and size of the chromosomes. However, there is a need in the art for improved methods of karyotyping, quickly and accurately.
The present invention provides methods for karyotyping. Preferably, the karotyping screens occur within droplets on a microfluidic device. Currently, scientists cannot accurately identify many translocations or other abnormalities using only a black and white karyotype. Spectral karyotyping (SKY) is a laboratory technique that allows scientists to visualize all 23 pairs of human chromosomes at one time, with each pair of chromosomes painted in a different fluorescent color. By using SKY, they can easily see instances where a chromosome, painted in one color, has a small piece of a different chromosome, painted in another color, attached to it.
Through the use of droplet-based methods, chromosomes can be captured within droplets without having to worry about shear-forces. The chromosomes can then be passed through a ‘neck down’ to stretch them out. Labeling prior to loading, with either Giemsa stain or oligonucleotide probes, can be used to karyotype the DNA as it flows.
The present invention provides methods of using SKY probes to ‘paint’ individual chromosomes. Also provided is a method used by flow cytometrists for the preparation of chromosomes prior to flow analysis, including flow sorting. The present invention provides methods which allow the adaptation of these methods for use on a microfluidic device.
SKY involves the preparation of a large collection of short sequences of single-stranded DNA called probes. Each of the individual probes in this DNA library is complementary to a unique region of one chromosome; together, all of the probes make up a network of DNA that is complementary to all of the chromosomes within the human genome. Each probe is labeled with a fluorescent molecule that corresponds to the chromosome to which it is complementary. For example, probes that are complementary to chromosome 1 are labeled with yellow molecules, while those that are complementary to chromosome 2 are labeled with red molecules, and so on. When these probes are mixed with the chromosomes from a human cell, the probes hybridize, or bind, to the DNA in the chromosomes. As they hybridize, the fluorescent probes essentially paint the set of chromosomes in a rainbow of colors. Scientists can then use computers to analyze the painted chromosomes to determine whether any of them exhibit translocations or other structural abnormalities. See, FIG. 35 .
Prior to analysis chromosomes can be prepared as described in Bee Ling Ng and Nigel P. Carter “Factors Affecting Flow Karyotype Resolution. Cytometry” Part A 69A:1028-1036 (2006) as follows:
    • 1. Arrest cells at metaphase using 0.11 g/ml demecolcine for optimal amount of time, dependent on the cell cycle time of the cell lines. (Approximately 5 h for suspension, 16 h for adherent cell lines and 4 h for LPS stimulated B lymphocyte culture).
    • 2. Harvest cells and centrifuge at 289 g for 5 min. Remove supernatant. 3. Resuspend cell pellet in 5 ml of hypotonic solution (75 mM KCl, 10 mM MgSO4, 0.2 mM spermine, 0.5 mM spermidine, pH 8.0) and incubate at room temperature for 10 min.
    • 4. Centrifuge cell suspension at 289 g for 5 min. Remove supernatant.
    • 5. Resuspend cell pellet in 3 ml of ice cold polyamine isolation buffer (PAB, containing 15 mM 20 Tris, 2 mM EDTA, 0.5 mM EGTA, 80 mM KCl 3 mM dithiothreitol, 0.25% Triton X-100, 0.2 mM spermine, 0.5 mM spermidine, pH 7.50) and vortex for 20 s.
    • 6. Briefly centrifuge chromosome suspensions at 201 g for 2 min. Filter supernatant through 201 m mesh filter.
    • 7. Stain chromosomes overnight with 51 g/ml of Hoechst, 401 g/ml chromomycin A3 and 10 mM MgSO4.
    • 8. To the stained chromosome suspension, add 10 mM of sodium citrate and 25 mM of sodium sulphite 1 h before flow analysis.
The present invention also provides methods of sorting chromosomes for karyotyping wherein individual chromosomes are sorted. This sorting can be performed after chromosome-specific identification (such as hybridization of labeled probes) so as to enrich a population for one or more specific chromosomes. This enriched population can be used in DNA sequencing reactions.
The Giemsa-stained and/or labeled-probe-hybridized chromosomes can be sent through a constriction of a channel on a microfluidic device to detect the areas of stain and/or label as a genetic ‘bar-code’ to identify regions of translocation, etc. on individual chromosomes.
Identification of chromosomes and karyotyping can be used after enrichment of specific cell-types, for example i) fetal cells from maternal blood, or ii) cancer cells from human blood.
Example 10
The present invention provides microbial strains with improved biomass conversion and methods of preparing such strains. Biomass is organic matter such as plant matter, i.e., trees, grasses, agricultural crops, or other biological material such as animal material. It can be used as a solid fuel, or converted into liquid or gaseous forms, for the production of electric power, heat, chemicals, or fuels. Biomass can also be used in formulating other commercial products in other industrial sectors such as textiles, food supply, environmental, communication, housing, etc. For example, biofuel development seeks the development of new microbial strains with improved biomass conversion to ethanol.
Researchers have been applying sophisticated metabolic engineering techniques to develop microorganisms that can more effectively ferment the sugars in biomass. Lignocellulosic biomass contains five carbon sugars such as xylose (from the hemicellulose) as well as the more “common” six carbon sugars such as glucose found in grains. This makes fermentation and other bioprocessing far more challenging. While some biorefinery scenarios will take advantage of the different sugar streams to produce multiple products, others will be more cost effective if all the sugars can coferment in a single set of equipment. Accordingly, researchers are developing microorganisms that can coferment all the sugars in biomass in order to improve ethanol production economics. With industrial partners, researchers are working to develop designer strains of microorganisms for biomass conversion of specific feedstocks, feedstreams, and processes. Thus, there is a need for devices and methods for the rapid engineering of new microbial strains with improved biomass conversion.
The present invention provides a microfluidic device in which to formulate a mutant bacterial, yeast, or fungi strain which can be used for biomass energy conversion. The microorganism strain can be engineered, e.g., by recombinant methods, to include at least one nucleic acid sequence encoding one or more polypeptides of interest, wherein the mutant strain expresses the polypeptides of interest at a higher level than the corresponding non-mutant strain under the same conditions.
In one embodiment, the nucleic acid sequence can be operably linked to an expression-regulating region selected from the group consisting of a promoter sequence associated with cellulase expression, xylanase expression, or gpdA expression. In another embodiment, the nucleic acid sequence can further be optionally linked to a secretion signal sequence.
In one embodiment, the nucleic acid sequence can be a heterologous nucleic acid sequence selected from heterologous polypeptide-encoding nucleic acid sequences, heterologous signal sequences, or heterologous expression-regulating sequences, or combinations thereof. For instance, the nucleic acid sequence can be a heterologous signal sequence, e.g., a secretion signal sequence. Alternatively, the nucleic acid sequence can be a heterologous expression-regulating region, e.g., an inducible promoter or a high expression promoter.
The polypeptides of interest can be homologous peptides and are expressed in the mutant strain at a higher level than in the corresponding non-mutant strain under the same conditions. In one embodiment, the polypeptides of interest can be selected from one or more of carbohydrate-degrading enzymes, proteases, lipases, esterases, other hydrolases, oxidoreductases, and transferases. In yet another embodiment, the polypeptides of interest can be selected from one or more of fungal enzymes that allow production or overproduction of primary metabolites, organic acids, secondary metabolites, and antibiotics. These fungal sequences can include secretion signal sequences, for example, and can be selected from one or more of cellulase, β-galactosidase, xylanase, pectinase, esterase, protease, amylase, polygalacturonase or hydrophobin. Alternatively, the fungal sequences can include one or more fungal expression-regulating regions. Preferably, the polypeptides of interest exhibit optimal activity and/or stability at a pH above 6, and/or have more than 70% of its activity and/or stability at a PH above 6.
The mutant microorganism can further include a selectable marker. The selectable marker can confer resistance to a drug, for example, or relieve a nutritional defect.
In another embodiment, the microorganism can be mutated via mutagenesis. Mutagenesis can be achieved, for example, by one or both of UV irradiation or chemical mutagenesis. For example, in one embodiment mutagenesis can include exposing a microorganism to UV irraditation, exposing it to N-methyl-N′-nitro-N-nitrosoguanidine, and exposing it again to UV irradiation.
The present invention also provides methods for creating microbial strains with improved biomass conversion. In one embodiment, the method includes providing a microfluidic device made of a microfabricated substrate. The microfabricated substrate can have a plurality of electrically addressable, channel bearing modules integrally arranged so as to be in fluid communication with each other, thereby forming at least one main channel adapted to carry at least one continuous phase fluid. The method further includes flowing a buffer, a microbe library, and a media (either in separate solutions or all together in one solution) through a first inlet channel into the main channel of the microfabricated substrate such that one or more droplets is formed in the continuous phase fluid; flowing a substrate through a second inlet channel into the main channel of the microfabricated substrate such that one or more droplets is formed in said continuous phase fluid; coalescing the droplets containing the microbe library with the droplets containing the substrate as the droplets pass through a coalescence module, thereby producing a NanoRefinery; interrogating the NanoRefinery for a predetermined characteristic within a detection module on the microfabricated substrate; and collecting the NanoRefineries containing the microbes of interest in a collection module on the microfabricated substrate. An assay system, e.g., a means by which to determine whether a desired product has been produced, or a means by which to determine the absence of a starting substrate material, can also be incorporated into one of the droplets or the NanoRefinery. The assay system can be added either before, after, or simultaneously with the addition of the substrate.
In one embodiment, the assay system is a dye that can measure the amount of sugar in a solution. In another embodiment, the microbe library can include one or more of DNA, bacteria, yeast or fungi. The substrate can include biomass, which can include one or more of fermentation broth, cellulose or other polysaccharide, or plant lignan.
The present invention further provides methods for degrading or converting biomass into one or more products. In one embodiment, the method includes treating the biomass with an effective amount of a recombinant microorganism, wherein the recombinant microorganism expresses or overexpresses one or more heterologous sequences encoding enzymes that degrade or convert the biomass into one or more products. In one embodiment, the biomass can include plant cell wall polysaccharides. In another embodiment, the products can include one or more of the commodity chemicals or secondary commodity chemicals used to produce one or more of the Intermediates or finished products and consumer goods listed in FIGS. 36 and 37 .
Example 11
The present invention provides methods of screening for enzymes with improved activity. As an example, at least one cell (prokaryotic or eukaryotic) is deposited into a droplet and the cells are allowed to secrete a substance for which a homogeneous assay is available. For a specific example, an individual Bacillus subtilis cell from a mutagen-treated culture is deposited within a 30 micron growth-medium containing droplet, the resulting droplets are collected and allowed to incubate overnight. The bacterium secretes a protease into the droplet. The droplets containing the Bacilli are then individually merged with an assay droplet containing a protease-cleavable dye-labeled peptide. The uncleaved peptide is colorless, while the cleaved peptide becomes red. The droplets are 15 incubated on chip for a sufficient time such as to allow color formation. Droplets that are red are sorted. The collected sorted droplets are then plated onto solid growth medium and the resulting colonies, after overnight incubation, represent individual clonal isolates from the droplets.
In addition, the assay droplets can be sorted based upon a specific activity, for example enzymes that are more active, indicated by a more intense red droplet after a specific period of time. In addition, the conditions within the droplet can be changed during the merging of the two droplets to assay conditions which may itself not be permissive for the bacteria. For example the pH within the droplet is altered in order to find mutant enzymes that work better under either acidic or alkaline conditions. Or the droplets can be heated such that enzymes that are more heat resistant are identified.
The merging of the droplets can be right after the individual cell is placed into the droplet or after further incubation.
Another method of the instant invention is as described above, except that the cells are lysed before the assay step in order to release the contents of the cell into the droplet.
In other embodiments, the substrate can be added with- or simultaneously formulated from two or more separate reagent streams with the contained within a droplet. For a specific example, macrophage cells are washed in buffer and incubated with an enzyme-conjugated anti-cell-surface antibody. The cells are then individually loaded into droplets formulated at the same time, with the enzyme substrate. The amount of enzyme substrate turned over within the droplet will be proportional to the number of enzyme molecules within the droplet, which is proportional to the number of antibodies bound to the macrophage surface. By careful calibration it should be possible to estimate the number of cell-surface molecules attached to the cell surface.

Claims (20)

What is claimed is:
1. A method for droplet formation for sample preparation for sequencing, the method comprising:
providing, via at least one inlet channel on a microfluidic substrate, an aqueous sample fluid comprising nucleic acid and at least one primer;
providing an immiscible first carrier fluid in a main channel on the microfluidic substrate;
producing a plurality of droplets comprising the aqueous sample fluid at a junction between the main channel and the at least one inlet channel;
copying at least a portion of the nucleic acid within one droplet of the plurality of droplets by extending the at least one primer to generate an extension product; and
breaking the plurality of droplets by introducing a droplet-destabilizing second fluid comprising a destabilizing surfactant, thereby recovering the aqueous sample fluid including the extension product from the plurality of droplets.
2. The method of claim 1, wherein the aqueous sample fluid comprises one or more cells.
3. The method of claim 1, further comprising subjecting the extension product to an amplification reaction.
4. The method of claim 3, wherein the amplification reaction is a PCR reaction.
5. The method of claim 1, wherein at least one oil supply line provides a constant flow of the first carrier fluid into a nozzle while the plurality of droplets are formed.
6. The method of claim 1, wherein the first carrier fluid comprises an oil.
7. The method of claim 6, wherein the oil comprises a fluorocarbon oil or a silicone oil.
8. The method of claim 6, wherein the oil comprises a surfactant.
9. The method of claim 8, wherein the surfactant is a fluorosurfactant.
10. The method of claim 1, wherein the microfluidic substrate is a microfluidic chip.
11. The method of claim 1, wherein the aqueous sample fluid comprises one or more beads.
12. The method of claim 1, further comprising centrifuging the plurality of droplets and the droplet-destabilizing second fluid to promote complete coalescence of the plurality of droplets.
13. The method of claim 1, wherein the destabilizing surfactant comprises a perfluorinated alcohol.
14. The method of claim 13, wherein the perfluorinated alcohol comprises 1H,1H,2H,2H-Perfluoro-1-octanol.
15. The method of claim 1, wherein the copying step uses a reverse transcriptase to produce complementary DNA.
16. A sample preparation method comprising:
flowing, via an inlet channel on a microfluidic substrate, an aqueous sample fluid comprising nucleic acid to a junction between the inlet channel and a main channel having an immiscible carrier fluid therein;
producing a plurality of droplets comprising the aqueous sample fluid surrounded by the immiscible carrier fluid in the main channel;
copying at least a portion of the nucleic acid within one droplet of the plurality of droplets by extending a primer to generate an extension product; and
recovering the aqueous sample fluid including the extension product from the plurality of droplets by introducing a droplet-destabilizing second fluid comprising a perfluorinated alcohol.
17. The method of claim 16, wherein the perfluorinated alcohol comprises 1H,1H,2H,2H-Perfluoro-1-octanol.
18. The method of claim 16, wherein the copying step uses a reverse transcriptase to produce complementary DNA.
19. The method of claim 16, wherein the aqueous fluid sample comprises one or more cells.
20. The method of claim 16, wherein the aqueous fluid sample comprises one or more beads.
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US6432290B1 (en) 1999-11-26 2002-08-13 The Governors Of The University Of Alberta Apparatus and method for trapping bead based reagents within microfluidic analysis systems
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JP2006507921A (en) 2002-06-28 2006-03-09 プレジデント・アンド・フェロウズ・オブ・ハーバード・カレッジ Method and apparatus for fluid dispersion
US11243494B2 (en) 2002-07-31 2022-02-08 Abs Global, Inc. Multiple laminar flow-based particle and cellular separation with laser steering
US7329545B2 (en) 2002-09-24 2008-02-12 Duke University Methods for sampling a liquid flow
US6911132B2 (en) 2002-09-24 2005-06-28 Duke University Apparatus for manipulating droplets by electrowetting-based techniques
US7445926B2 (en) * 2002-12-30 2008-11-04 The Regents Of The University Of California Fluid control structures in microfluidic devices
IL154677A0 (en) * 2003-02-27 2003-09-17 Univ Bar Ilan A method and apparatus for manipulating an individual cell
US20060078893A1 (en) 2004-10-12 2006-04-13 Medical Research Council Compartmentalised combinatorial chemistry by microfluidic control
GB0307403D0 (en) 2003-03-31 2003-05-07 Medical Res Council Selection by compartmentalised screening
GB0307428D0 (en) 2003-03-31 2003-05-07 Medical Res Council Compartmentalised combinatorial chemistry
EP2266687A3 (en) 2003-04-10 2011-06-29 The President and Fellows of Harvard College Formation and control of fluidic species
FR2856498B1 (en) * 2003-06-19 2005-09-30 Goulven Jean Alain Vernois DISTRIBUTION UNDER CONTROLLED ATMOSPHERE
US9200245B2 (en) * 2003-06-26 2015-12-01 Seng Enterprises Ltd. Multiwell plate
US7888110B2 (en) * 2003-06-26 2011-02-15 Seng Enterprises Ltd. Pico liter well holding device and method of making the same
US8597597B2 (en) * 2003-06-26 2013-12-03 Seng Enterprises Ltd. Picoliter well holding device and method of making the same
CN1842368B (en) 2003-08-27 2014-05-28 哈佛大学 Electronic control of fluid species
US9597644B2 (en) 2003-09-05 2017-03-21 Stokes Bio Limited Methods for culturing and analyzing cells
EP1663497B2 (en) 2003-09-05 2020-03-25 Stokes Bio Limited A microfluidic analysis system
GB0329220D0 (en) * 2003-12-17 2004-01-21 Inverness Medical Switzerland System
US20050221339A1 (en) * 2004-03-31 2005-10-06 Medical Research Council Harvard University Compartmentalised screening by microfluidic control
US7799553B2 (en) 2004-06-01 2010-09-21 The Regents Of The University Of California Microfabricated integrated DNA analysis system
US7692219B1 (en) 2004-06-25 2010-04-06 University Of Hawaii Ultrasensitive biosensors
US7655470B2 (en) 2004-10-29 2010-02-02 University Of Chicago Method for manipulating a plurality of plugs and performing reactions therein in microfluidic systems
US9477233B2 (en) 2004-07-02 2016-10-25 The University Of Chicago Microfluidic system with a plurality of sequential T-junctions for performing reactions in microdroplets
WO2006003664A1 (en) * 2004-07-07 2006-01-12 Seng Enterprises Ltd. Method and device for identifying an image of a well in an image of a well-bearing component
US20080009051A1 (en) * 2004-08-25 2008-01-10 Seng Enterprises Ltd. Method and Device for Isolating Cells
CN102759466A (en) 2004-09-15 2012-10-31 英特基因有限公司 Microfluidic devices
US7745207B2 (en) 2006-02-03 2010-06-29 IntegenX, Inc. Microfluidic devices
US7968287B2 (en) 2004-10-08 2011-06-28 Medical Research Council Harvard University In vitro evolution in microfluidic systems
ES2352344T3 (en) 2005-01-25 2011-02-17 Seng Enterprises Limited MICROFLUID DEVICE FOR CELL STUDY.
ES2390800T3 (en) 2005-01-28 2012-11-16 Duke University Apparatus and methods for handling droplets on a printed circuit board
JP2008535644A (en) 2005-03-04 2008-09-04 プレジデント・アンド・フエローズ・オブ・ハーバード・カレツジ Method and apparatus for the formation of multiple emulsions
US20070054119A1 (en) * 2005-03-04 2007-03-08 Piotr Garstecki Systems and methods of forming particles
US8153435B1 (en) 2005-03-30 2012-04-10 Tracer Detection Technology Corp. Methods and articles for identifying objects using encapsulated perfluorocarbon tracers
CA2611671C (en) 2005-06-15 2013-10-08 Callida Genomics, Inc. Single molecule arrays for genetic and chemical analysis
US7928967B2 (en) * 2005-08-19 2011-04-19 Silverbrook Research Pty Ltd Force sensor with angled coupling
US20070044824A1 (en) * 2005-09-01 2007-03-01 Scott William Capeci Processing system and method of processing
US20070141555A1 (en) * 2005-10-11 2007-06-21 Mordechai Deutsch Current damper for the study of cells
WO2007052245A1 (en) * 2005-11-03 2007-05-10 Seng Enterprises Ltd. Method and device for studying floating, living cells
EP2363205A3 (en) 2006-01-11 2014-06-04 Raindance Technologies, Inc. Microfluidic Devices And Methods Of Use In The Formation And Control Of Nanoreactors
WO2007089541A2 (en) * 2006-01-27 2007-08-09 President And Fellows Of Harvard College Fluidic droplet coalescence
US8298833B2 (en) 2006-02-07 2012-10-30 Stokes Bio Limited Liquid bridge and system
US7766033B2 (en) 2006-03-22 2010-08-03 The Regents Of The University Of California Multiplexed latching valves for microfluidic devices and processors
US9476856B2 (en) 2006-04-13 2016-10-25 Advanced Liquid Logic, Inc. Droplet-based affinity assays
US8637317B2 (en) 2006-04-18 2014-01-28 Advanced Liquid Logic, Inc. Method of washing beads
US20140193807A1 (en) 2006-04-18 2014-07-10 Advanced Liquid Logic, Inc. Bead manipulation techniques
US8613889B2 (en) 2006-04-13 2013-12-24 Advanced Liquid Logic, Inc. Droplet-based washing
US8492168B2 (en) 2006-04-18 2013-07-23 Advanced Liquid Logic Inc. Droplet-based affinity assays
US8980198B2 (en) * 2006-04-18 2015-03-17 Advanced Liquid Logic, Inc. Filler fluids for droplet operations
US8637324B2 (en) 2006-04-18 2014-01-28 Advanced Liquid Logic, Inc. Bead incubation and washing on a droplet actuator
US7901947B2 (en) 2006-04-18 2011-03-08 Advanced Liquid Logic, Inc. Droplet-based particle sorting
US8716015B2 (en) 2006-04-18 2014-05-06 Advanced Liquid Logic, Inc. Manipulation of cells on a droplet actuator
US7763471B2 (en) 2006-04-18 2010-07-27 Advanced Liquid Logic, Inc. Method of electrowetting droplet operations for protein crystallization
US8658111B2 (en) 2006-04-18 2014-02-25 Advanced Liquid Logic, Inc. Droplet actuators, modified fluids and methods
US10078078B2 (en) 2006-04-18 2018-09-18 Advanced Liquid Logic, Inc. Bead incubation and washing on a droplet actuator
US7816121B2 (en) 2006-04-18 2010-10-19 Advanced Liquid Logic, Inc. Droplet actuation system and method
US7851184B2 (en) 2006-04-18 2010-12-14 Advanced Liquid Logic, Inc. Droplet-based nucleic acid amplification method and apparatus
US7439014B2 (en) 2006-04-18 2008-10-21 Advanced Liquid Logic, Inc. Droplet-based surface modification and washing
US7815871B2 (en) 2006-04-18 2010-10-19 Advanced Liquid Logic, Inc. Droplet microactuator system
WO2007123908A2 (en) 2006-04-18 2007-11-01 Advanced Liquid Logic, Inc. Droplet-based multiwell operations
US8809068B2 (en) 2006-04-18 2014-08-19 Advanced Liquid Logic, Inc. Manipulation of beads in droplets and methods for manipulating droplets
US8470606B2 (en) 2006-04-18 2013-06-25 Duke University Manipulation of beads in droplets and methods for splitting droplets
US8041463B2 (en) 2006-05-09 2011-10-18 Advanced Liquid Logic, Inc. Modular droplet actuator drive
US9675972B2 (en) 2006-05-09 2017-06-13 Advanced Liquid Logic, Inc. Method of concentrating beads in a droplet
US7939021B2 (en) 2007-05-09 2011-05-10 Advanced Liquid Logic, Inc. Droplet actuator analyzer with cartridge
US7822510B2 (en) 2006-05-09 2010-10-26 Advanced Liquid Logic, Inc. Systems, methods, and products for graphically illustrating and controlling a droplet actuator
EP3782722B1 (en) * 2006-05-11 2022-07-06 Bio-Rad Laboratories, Inc. Microfluidic devices
US9562837B2 (en) 2006-05-11 2017-02-07 Raindance Technologies, Inc. Systems for handling microfludic droplets
EP3536396B1 (en) 2006-08-07 2022-03-30 The President and Fellows of Harvard College Fluorocarbon emulsion stabilizing surfactants
JP4968896B2 (en) * 2006-09-27 2012-07-04 富士フイルム株式会社 Dispersion manufacturing apparatus and dispersion manufacturing method
US8550503B2 (en) * 2006-09-28 2013-10-08 Stokes Bio Ltd. Microfluidic connector
US20100297748A1 (en) * 2006-09-28 2010-11-25 Stokes Bio Limited Integrated fluidic circuits
WO2008052138A2 (en) 2006-10-25 2008-05-02 The Regents Of The University Of California Inline-injection microdevice and microfabricated integrated dna analysis system using same
US8349167B2 (en) 2006-12-14 2013-01-08 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
JP5622392B2 (en) 2006-12-14 2014-11-12 ライフ テクノロジーズ コーポレーション Method and apparatus for analyte measurement using large-scale FET arrays
US11339430B2 (en) 2007-07-10 2022-05-24 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8262900B2 (en) * 2006-12-14 2012-09-11 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US7696002B1 (en) * 2007-01-25 2010-04-13 Pacesette, Inc. Electrolytic capacitor case design with insert molded elastomer seals
GB0701641D0 (en) * 2007-01-29 2007-03-07 Iti Scotland Ltd Analyte manipulation and detection
US8753894B2 (en) * 2007-02-01 2014-06-17 Diagnostic Biosensors, Llc Integrated membrane sensor
KR20100028526A (en) 2007-02-05 2010-03-12 마이크로칩 바이오테크놀로지스, 인크. Microfluidic and nanofluidic devices, systems, and applications
US8772046B2 (en) * 2007-02-06 2014-07-08 Brandeis University Manipulation of fluids and reactions in microfluidic systems
KR101503510B1 (en) 2007-02-09 2015-03-18 어드밴스드 리퀴드 로직, 아이엔씨. Droplet actuator devices and methods employing magnetic beads
US9029085B2 (en) 2007-03-07 2015-05-12 President And Fellows Of Harvard College Assays and other reactions involving droplets
WO2008121342A2 (en) * 2007-03-28 2008-10-09 President And Fellows Of Harvard College Emulsions and techniques for formation
JP2008245612A (en) * 2007-03-30 2008-10-16 Hitachi Ltd Sample preparation method and apparatus
US8592221B2 (en) 2007-04-19 2013-11-26 Brandeis University Manipulation of fluids, fluid components and reactions in microfluidic systems
US8691164B2 (en) * 2007-04-20 2014-04-08 Celula, Inc. Cell sorting system and methods
US20080268440A1 (en) * 2007-04-26 2008-10-30 Liu Timothy Z Biomolecule immobilization on surface via hydrophobic interactions
CN103157400B (en) * 2007-06-21 2014-12-24 简·探针公司 Instrument and method for exposing container to multiple areas
US8454906B2 (en) * 2007-07-24 2013-06-04 The Regents Of The University Of California Microfabricated droplet generator for single molecule/cell genetic analysis in engineered monodispersed emulsions
WO2009015390A2 (en) * 2007-07-26 2009-01-29 University Of Chicago Co-incuating confined microbial communities
CN104307581B (en) * 2007-08-09 2017-04-12 诺思可有限公司 Methods and devices for correlated, multi-parameter single cell measurements and recovery of remnant biological material
US8268246B2 (en) 2007-08-09 2012-09-18 Advanced Liquid Logic Inc PCB droplet actuator fabrication
WO2009032863A2 (en) 2007-09-04 2009-03-12 Advanced Liquid Logic, Inc. Droplet actuator with improved top substrate
US9744513B2 (en) 2007-09-20 2017-08-29 Jean-Louis Viovy Encapsulation microfluidic device
US8268564B2 (en) * 2007-09-26 2012-09-18 President And Fellows Of Harvard College Methods and applications for stitched DNA barcodes
GB0720202D0 (en) 2007-10-16 2007-11-28 Cambridge Entpr Ltd Microfluidic systems
US9267918B2 (en) 2007-10-16 2016-02-23 Cambridge Enterprise Limited Microfluidic systems
US9114397B2 (en) * 2007-10-25 2015-08-25 Canon U.S. Life Sciences, Inc. Method of reducing cross-contamination in continuous amplification reactions in a channel
US9145540B1 (en) 2007-11-15 2015-09-29 Seng Enterprises Ltd. Device for the study of living cells
US8592150B2 (en) 2007-12-05 2013-11-26 Complete Genomics, Inc. Methods and compositions for long fragment read sequencing
US9797010B2 (en) 2007-12-21 2017-10-24 President And Fellows Of Harvard College Systems and methods for nucleic acid sequencing
BRPI0821734A2 (en) 2007-12-23 2022-10-25 Advanced Liquid Logic Inc DROPLET AUTHOR SETTINGS AND METHODS FOR CONDUCTING DROPLET OPERATIONS.
EP2237887A2 (en) * 2007-12-26 2010-10-13 Seng Enterprises Ltd. Device for the study of living cells
US8815576B2 (en) * 2007-12-27 2014-08-26 Lawrence Livermore National Security, Llc. Chip-based sequencing nucleic acids
US8969257B1 (en) * 2007-12-28 2015-03-03 Intermolecular, Inc. Combinatorial flow system and method
WO2009108260A2 (en) 2008-01-22 2009-09-03 Microchip Biotechnologies, Inc. Universal sample preparation system and use in an integrated analysis system
CN102015998A (en) * 2008-02-01 2011-04-13 加利福尼亚大学董事会 Microfluidic imaging cytometry
US8062903B2 (en) * 2008-03-03 2011-11-22 University Of Washington Droplet compartmentalization for chemical separation and on-line sampling
US9409177B2 (en) * 2008-03-21 2016-08-09 Lawrence Livermore National Security, Llc Chip-based device for parallel sorting, amplification, detection, and identification of nucleic acid subsequences
WO2009134395A2 (en) 2008-04-28 2009-11-05 President And Fellows Of Harvard College Microfluidic device for storage and well-defined arrangement of droplets
WO2009137415A2 (en) 2008-05-03 2009-11-12 Advanced Liquid Logic, Inc. Reagent and sample preparation, loading, and storage
US20100075436A1 (en) * 2008-05-06 2010-03-25 Urdea Michael S Methods for use with nanoreactors
EP2291509B1 (en) * 2008-05-16 2023-11-15 Board of Supervisors of Louisiana State University and Agricultural and Mechanical College Microfluidic isolation of tumor cells or other rare cells from whole blood or other liquids
US7900503B2 (en) * 2008-05-17 2011-03-08 William Keith Leonard Method of high throughput viscometry
US8622987B2 (en) 2008-06-04 2014-01-07 The University Of Chicago Chemistrode, a plug-based microfluidic device and method for stimulation and sampling with high temporal, spatial, and chemical resolution
EP2307577B1 (en) 2008-06-25 2015-06-03 Life Technologies Corporation Methods for measuring analytes using large scale fet arrays
WO2010009365A1 (en) 2008-07-18 2010-01-21 Raindance Technologies, Inc. Droplet libraries
US12038438B2 (en) 2008-07-18 2024-07-16 Bio-Rad Laboratories, Inc. Enzyme quantification
US9180453B2 (en) * 2008-08-15 2015-11-10 University Of Washington Method and apparatus for the discretization and manipulation of sample volumes
DE102008039117B3 (en) 2008-08-21 2010-05-20 Institut für Bioprozess- und Analysenmesstechnik e.V. Arrangement and method for generating, manipulating and analyzing compartments
DK2315841T3 (en) 2008-08-21 2012-11-19 Novozymes As Method of screening in a microfluidic device
US8583380B2 (en) 2008-09-05 2013-11-12 Aueon, Inc. Methods for stratifying and annotating cancer drug treatment options
WO2010033200A2 (en) 2008-09-19 2010-03-25 President And Fellows Of Harvard College Creation of libraries of droplets and related species
US8709762B2 (en) 2010-03-02 2014-04-29 Bio-Rad Laboratories, Inc. System for hot-start amplification via a multiple emulsion
US9156010B2 (en) 2008-09-23 2015-10-13 Bio-Rad Laboratories, Inc. Droplet-based assay system
US8951939B2 (en) 2011-07-12 2015-02-10 Bio-Rad Laboratories, Inc. Digital assays with multiplexed detection of two or more targets in the same optical channel
US9764322B2 (en) 2008-09-23 2017-09-19 Bio-Rad Laboratories, Inc. System for generating droplets with pressure monitoring
US9417190B2 (en) 2008-09-23 2016-08-16 Bio-Rad Laboratories, Inc. Calibrations and controls for droplet-based assays
US8633015B2 (en) 2008-09-23 2014-01-21 Bio-Rad Laboratories, Inc. Flow-based thermocycling system with thermoelectric cooler
US9194861B2 (en) 2009-09-02 2015-11-24 Bio-Rad Laboratories, Inc. Method of mixing fluids by coalescence of multiple emulsions
US10512910B2 (en) 2008-09-23 2019-12-24 Bio-Rad Laboratories, Inc. Droplet-based analysis method
WO2011120020A1 (en) 2010-03-25 2011-09-29 Quantalife, Inc. Droplet transport system for detection
US8179032B2 (en) * 2008-09-23 2012-05-15 The Board Of Trustees Of The University Of Illinois Ellipsoidal microcavity plasma devices and powder blasting formation
US11130128B2 (en) 2008-09-23 2021-09-28 Bio-Rad Laboratories, Inc. Detection method for a target nucleic acid
US12162008B2 (en) 2008-09-23 2024-12-10 Bio-Rad Laboratories, Inc. Partition-based method of analysis
US9132394B2 (en) 2008-09-23 2015-09-15 Bio-Rad Laboratories, Inc. System for detection of spaced droplets
US12090480B2 (en) 2008-09-23 2024-09-17 Bio-Rad Laboratories, Inc. Partition-based method of analysis
CA3149293C (en) * 2008-09-23 2023-09-12 Bio-Rad Laboratories, Inc. Droplet-based assay system
US9492797B2 (en) 2008-09-23 2016-11-15 Bio-Rad Laboratories, Inc. System for detection of spaced droplets
WO2010042744A1 (en) * 2008-10-08 2010-04-15 Universite De Strasbourg Microfluidic devices for reliable on-chip incubation of droplets in delay lines
GB0818609D0 (en) 2008-10-10 2008-11-19 Univ Hull apparatus and method
US20100301398A1 (en) 2009-05-29 2010-12-02 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
US20100137143A1 (en) 2008-10-22 2010-06-03 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
WO2010053796A2 (en) * 2008-10-28 2010-05-14 The Regents Of The University Of Colorado, A Body Corporate Microfluidic cell sorter utilizing broadband coherent anti-stokes raman scattering
US9057568B2 (en) 2008-12-16 2015-06-16 California Institute Of Technology Temperature control devices and methods
EP2373812B1 (en) * 2008-12-19 2016-11-09 President and Fellows of Harvard College Particle-assisted nucleic acid sequencing
EP2384429A1 (en) * 2008-12-31 2011-11-09 Integenx Inc. Instrument with microfluidic chip
US20130034863A1 (en) * 2009-01-23 2013-02-07 Philadelphia Health And Education Corporation Apparatus and Methods for Detecting Inflammation Using Quantum Dots
KR101793744B1 (en) 2009-03-13 2017-11-03 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 Scale-up of flow-focusing microfluidic devices
EP3415235B1 (en) 2009-03-23 2025-11-12 Bio-Rad Laboratories, Inc. Manipulation of microfluidic droplets
US9447461B2 (en) 2009-03-24 2016-09-20 California Institute Of Technology Analysis devices, kits, and related methods for digital quantification of nucleic acids and other analytes
JP5766178B2 (en) 2009-03-24 2015-08-19 ザ・ユニバーシティ・オブ・シカゴＴｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｃｈｉｃａｇｏ Slipchip apparatus and method
US9464319B2 (en) 2009-03-24 2016-10-11 California Institute Of Technology Multivolume devices, kits and related methods for quantification of nucleic acids and other analytes
US10196700B2 (en) 2009-03-24 2019-02-05 University Of Chicago Multivolume devices, kits and related methods for quantification and detection of nucleic acids and other analytes
ES2638861T3 (en) * 2009-04-13 2017-10-24 University Of Washington Rare particle detection method and apparatus
JP5881936B2 (en) * 2009-04-20 2016-03-09 ソニー株式会社 Sample solution introduction kit and sample solution injector
EP2425240A4 (en) 2009-04-30 2012-12-12 Good Start Genetics Inc Methods and compositions for evaluating genetic markers
US12129514B2 (en) 2009-04-30 2024-10-29 Molecular Loop Biosolutions, Llc Methods and compositions for evaluating genetic markers
DE102009040151B4 (en) * 2009-05-26 2013-09-12 Analytik Jena Ag Arrangement for the detection of chemiluminescence on gases
WO2010138187A1 (en) * 2009-05-29 2010-12-02 Ion Torrent Systems Incorporated Scaffolded nucleic acid polymer particles and methods of making and using
US20120261274A1 (en) 2009-05-29 2012-10-18 Life Technologies Corporation Methods and apparatus for measuring analytes
US8673627B2 (en) 2009-05-29 2014-03-18 Life Technologies Corporation Apparatus and methods for performing electrochemical reactions
US8574835B2 (en) 2009-05-29 2013-11-05 Life Technologies Corporation Scaffolded nucleic acid polymer particles and methods of making and using
US8776573B2 (en) 2009-05-29 2014-07-15 Life Technologies Corporation Methods and apparatus for measuring analytes
WO2010141326A1 (en) * 2009-06-02 2010-12-09 Integenx Inc. Fluidic devices with diaphragm valves
CA2764678C (en) * 2009-06-04 2017-12-12 Lockheed Martin Corporation Multiple-sample microfluidic chip for dna analysis
WO2010141921A1 (en) * 2009-06-05 2010-12-09 Integenx Inc. Universal sample preparation system and use in an integrated analysis system
US9524369B2 (en) 2009-06-15 2016-12-20 Complete Genomics, Inc. Processing and analysis of complex nucleic acid sequence data
EP2977455B1 (en) * 2009-06-15 2020-04-15 Complete Genomics, Inc. Method for long fragment read sequencing
WO2010147942A1 (en) * 2009-06-16 2010-12-23 Massachusetts Institute Of Technology Multiphase non-linear electrokinetic devices
WO2010151776A2 (en) * 2009-06-26 2010-12-29 President And Fellows Of Harvard College Fluid injection
FR2948024B1 (en) * 2009-07-17 2020-01-10 Centre National De La Recherche Scientifique - Cnrs - ULTRASOUND ACTIVABLE EMULSION AND MANUFACTURING METHOD THEREOF.
WO2011011172A1 (en) * 2009-07-21 2011-01-27 IntegenX, Inc. Microfluidic devices and uses thereof
EP2459750A1 (en) * 2009-07-29 2012-06-06 Pyrobett Pte Ltd Method and apparatus for conducting an assay
US9063136B2 (en) * 2009-08-02 2015-06-23 Qvella Corporation Cell concentration, capture and lysis devices and methods of use thereof
CN102612652B (en) * 2009-08-06 2016-05-04 康奈尔大学 For equipment and the method for analysis of molecules
US8202486B2 (en) * 2009-08-12 2012-06-19 Caliper Life Sciences, Inc. Pinching channels for fractionation of fragmented samples
US8926065B2 (en) 2009-08-14 2015-01-06 Advanced Liquid Logic, Inc. Droplet actuator devices and methods
US20120211084A1 (en) 2009-09-02 2012-08-23 President And Fellows Of Harvard College Multiple emulsions created using jetting and other techniques
EP2486409A1 (en) 2009-10-09 2012-08-15 Universite De Strasbourg Labelled silica-based nanomaterial with enhanced properties and uses thereof
US8188438B2 (en) * 2009-10-20 2012-05-29 Diagnostics Chips, LLC Electrokinetic microfluidic flow cytometer apparatuses with differential resistive particle counting and optical sorting
EP3842150A1 (en) * 2009-10-27 2021-06-30 President and Fellows of Harvard College Droplet creation techniques
EP2494353B1 (en) 2009-10-30 2017-09-27 Illumina Inc. An apparatus comprising a plurality of encoded microvessels and a plurality of compartments and a method of reading a plurality of encoded micro vessels
US10207240B2 (en) 2009-11-03 2019-02-19 Gen9, Inc. Methods and microfluidic devices for the manipulation of droplets in high fidelity polynucleotide assembly
WO2011057197A2 (en) 2009-11-06 2011-05-12 Advanced Liquid Logic, Inc. Integrated droplet actuator for gel electrophoresis and molecular analysis
CA2767028A1 (en) * 2009-11-25 2011-06-03 Quantalife, Inc. Methods and compositions for detecting genetic material
WO2011066185A1 (en) 2009-11-25 2011-06-03 Gen9, Inc. Microfluidic devices and methods for gene synthesis
JP2013511991A (en) * 2009-11-25 2013-04-11 クアンタライフ， インコーポレイテッド Methods and compositions for detecting genetic material
US8584703B2 (en) * 2009-12-01 2013-11-19 Integenx Inc. Device with diaphragm valve
CA2781581C (en) * 2009-12-01 2018-03-06 Oxford Nanopore Technologies Limited Biochemical analysis instrument
EP2516669B1 (en) 2009-12-21 2016-10-12 Advanced Liquid Logic, Inc. Enzyme assays on a droplet actuator
WO2011079176A2 (en) 2009-12-23 2011-06-30 Raindance Technologies, Inc. Microfluidic systems and methods for reducing the exchange of molecules between droplets
WO2011085075A2 (en) * 2010-01-07 2011-07-14 Gen9, Inc. Assembly of high fidelity polynucleotides
JP5934657B2 (en) 2010-02-12 2016-06-15 レインダンス テクノロジーズ， インコーポレイテッド Digital specimen analysis
US10351905B2 (en) 2010-02-12 2019-07-16 Bio-Rad Laboratories, Inc. Digital analyte analysis
US9366632B2 (en) 2010-02-12 2016-06-14 Raindance Technologies, Inc. Digital analyte analysis
US9494520B2 (en) * 2010-02-12 2016-11-15 Raindance Technologies, Inc. Digital analyte analysis
US9399797B2 (en) 2010-02-12 2016-07-26 Raindance Technologies, Inc. Digital analyte analysis
US8399198B2 (en) 2010-03-02 2013-03-19 Bio-Rad Laboratories, Inc. Assays with droplets transformed into capsules
US20130052648A1 (en) * 2010-03-12 2013-02-28 The General Hospital Corporation Amplifying rare cell surface markers
AR080405A1 (en) * 2010-03-17 2012-04-04 Basf Se EMULSIFICATION TO FOUND
EP2550351A4 (en) 2010-03-25 2014-07-09 Quantalife Inc Detection system for droplet-based assays
CA2767182C (en) 2010-03-25 2020-03-24 Bio-Rad Laboratories, Inc. Droplet generation for droplet-based assays
FR2958186A1 (en) * 2010-03-30 2011-10-07 Ecole Polytech DEVICE FOR FORMING DROPS IN A MICROFLUID CIRCUIT.
ITTO20100068U1 (en) * 2010-04-20 2011-10-21 Eltek Spa MICROFLUID AND / OR EQUIPMENT DEVICES FOR MICROFLUID DEVICES
US9442106B2 (en) * 2010-05-08 2016-09-13 Universiteit Twente Simple and affordable method for immunophenotyping using a microfluidic chip sample preparation with image cytometry
US8512538B2 (en) 2010-05-28 2013-08-20 Integenx Inc. Capillary electrophoresis device
US20120001646A1 (en) 2010-06-30 2012-01-05 Life Technologies Corporation Methods and apparatus for testing isfet arrays
CN103154718B (en) 2010-06-30 2015-09-23 生命科技公司 Charge accumulation circuits and methods for sensing ions
CN119165030A (en) 2010-06-30 2024-12-20 生命科技公司 Array Integrator
US11307166B2 (en) 2010-07-01 2022-04-19 Life Technologies Corporation Column ADC
CN103168341B (en) 2010-07-03 2016-10-05 生命科技公司 There is the chemosensitive sensor of lightly doped discharger
DE102010032203A1 (en) * 2010-07-26 2012-01-26 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Method and apparatus for the passive separation and sorting of drops, in particular in a microfluidic system, by using non-optical markers for reactions within the drops
US8968873B2 (en) * 2010-08-13 2015-03-03 University Of Connecticut Co-flow microfluidic device for polymersome formation
WO2012024658A2 (en) 2010-08-20 2012-02-23 IntegenX, Inc. Integrated analysis system
EP2606242A4 (en) 2010-08-20 2016-07-20 Integenx Inc MICROFLUIDIC DEVICES HAVING MECHANICALLY SEALED DIAPHRAGM VALVES
WO2012030878A1 (en) 2010-08-31 2012-03-08 Canon U.S. Life Sciences, Inc. Composition and method for in-system priming microfluidic devices
US9114399B2 (en) 2010-08-31 2015-08-25 Canon U.S. Life Sciences, Inc. System and method for serial processing of multiple nucleic acid assays
WO2012036679A1 (en) 2010-09-15 2012-03-22 Life Technologies Corporation Methods and apparatus for measuring analytes
US8796036B2 (en) 2010-09-24 2014-08-05 Life Technologies Corporation Method and system for delta double sampling
US9309556B2 (en) 2010-09-24 2016-04-12 The Board Of Trustees Of The Leland Stanford Junior University Direct capture, amplification and sequencing of target DNA using immobilized primers
US9562897B2 (en) 2010-09-30 2017-02-07 Raindance Technologies, Inc. Sandwich assays in droplets
US8486703B2 (en) 2010-09-30 2013-07-16 Ut-Battelle, Llc Surface sampling concentration and reaction probe
EP2436444A1 (en) * 2010-10-01 2012-04-04 Centre National de la Recherche Scientifique (C.N.R.S.) Microfluidic device for production and collection of droplets of a fluid
MX2013004184A (en) 2010-10-15 2013-07-29 Lockheed Corp Micro fluidic optic design.
WO2012061444A2 (en) 2010-11-01 2012-05-10 Hiddessen Amy L System for forming emulsions
US10908066B2 (en) 2010-11-16 2021-02-02 1087 Systems, Inc. Use of vibrational spectroscopy for microfluidic liquid measurement
JP6096672B2 (en) 2010-12-07 2017-03-15 ヌビオ，インコーポレイテッド Nucleic acid target detection using detectors, probes, and inhibitors
DE102010054879B4 (en) 2010-12-17 2013-07-18 Institut für Bioprozess- und Analysenmesstechnik e.V. Arrangement and method for conditioning fluid compartments
US20120167410A1 (en) * 2010-12-21 2012-07-05 Basf Se Spray drying techniques
US9233369B2 (en) 2010-12-23 2016-01-12 California Institute Of Technology Fluidic devices and fabrication methods for microfluidics
US8968585B2 (en) * 2010-12-23 2015-03-03 California Institute Of Technology Methods of fabrication of cartridges for biological analysis
US9163281B2 (en) 2010-12-23 2015-10-20 Good Start Genetics, Inc. Methods for maintaining the integrity and identification of a nucleic acid template in a multiplex sequencing reaction
US8871500B2 (en) 2011-01-21 2014-10-28 Innovative Micro Technology MEMS particle sorting actuator and method of manufacturing
JP2014505594A (en) * 2011-02-07 2014-03-06 プレジデント アンド フェロウズ オブ ハーバード カレッジ System and method for splitting droplets
CA2826748C (en) 2011-02-09 2020-08-04 Bio-Rad Laboratories, Inc. Method of detecting variations in copy number of a target nucleic acid
US9266104B2 (en) 2011-02-11 2016-02-23 Raindance Technologies, Inc. Thermocycling device for nucleic acid amplification and methods of use
EP3859011A1 (en) 2011-02-11 2021-08-04 Bio-Rad Laboratories, Inc. Methods for forming mixed droplets
DK2675899T1 (en) * 2011-02-16 2016-08-22 Glycosyn LLC Biosynthesis of human milk oligosaccharides in the engineered bacteria
US12097495B2 (en) 2011-02-18 2024-09-24 Bio-Rad Laboratories, Inc. Methods and compositions for detecting genetic material
EP2675819B1 (en) 2011-02-18 2020-04-08 Bio-Rad Laboratories, Inc. Compositions and methods for molecular labeling
EP2686449B1 (en) 2011-03-18 2020-11-18 Bio-Rad Laboratories, Inc. Multiplexed digital assays with combinatorial use of signals
SG193437A1 (en) 2011-03-31 2013-10-30 Gnubio Inc Scalable spectroscopic detection and measurement
WO2012135664A2 (en) 2011-03-31 2012-10-04 Dana-Farber Cancer Institute, Inc. Methods and compositions to enable multiplex cold-pcr
EP2694982A1 (en) * 2011-04-08 2014-02-12 Stokes Bio Limited Systems and methods for continuous flow pcr systems
KR101356279B1 (en) 2011-04-21 2014-01-28 충북대학교 산학협력단 System and method for detecting interaction between biomolecules
CA2834291A1 (en) 2011-04-25 2012-11-01 Biorad Laboratories, Inc. Methods and compositions for nucleic acid analysis
US9188615B2 (en) 2011-05-09 2015-11-17 Advanced Liquid Logic, Inc. Microfluidic feedback using impedance detection
BR112013029729A2 (en) 2011-05-23 2017-01-24 Basf Se emulsion control including multiple emulsions
US8841071B2 (en) 2011-06-02 2014-09-23 Raindance Technologies, Inc. Sample multiplexing
EP3216872B1 (en) 2011-06-02 2020-04-01 Bio-Rad Laboratories, Inc. Enzyme quantification
WO2012174142A1 (en) 2011-06-17 2012-12-20 Life Technologies Corporation Flat-field imaging system and methods of use
US20140220350A1 (en) 2011-07-06 2014-08-07 President And Fellows Of Harvard College Multiple emulsions and techniques for the formation of multiple emulsions
JP5894272B2 (en) 2011-07-06 2016-03-23 アドバンスト リキッド ロジック インコーポレイテッドＡｄｖａｎｃｅｄ Ｌｉｑｕｉｄ Ｌｏｇｉｃ， Ｉｎｃ． Reagent storage in droplet actuators
US9513253B2 (en) 2011-07-11 2016-12-06 Advanced Liquid Logic, Inc. Droplet actuators and techniques for droplet-based enzymatic assays
US8658430B2 (en) * 2011-07-20 2014-02-25 Raindance Technologies, Inc. Manipulating droplet size
JP6212488B2 (en) 2011-07-25 2017-10-11 クヴェッラ コーポレーション Methods and devices for electrical sample preparation
WO2013016413A2 (en) 2011-07-25 2013-01-31 Advanced Liquid Logic Inc Droplet actuator apparatus and system
WO2013019751A1 (en) 2011-07-29 2013-02-07 Bio-Rad Laboratories, Inc., Library characterization by digital assay
US10466160B2 (en) 2011-08-01 2019-11-05 Celsee Diagnostics, Inc. System and method for retrieving and analyzing particles
WO2013019491A1 (en) 2011-08-01 2013-02-07 Denovo Sciences Cell capture system and method of use
US9174216B2 (en) 2013-03-13 2015-11-03 DeNovo Science, Inc. System for capturing and analyzing cells
US9404864B2 (en) 2013-03-13 2016-08-02 Denovo Sciences, Inc. System for imaging captured cells
US20150205272A1 (en) * 2011-08-05 2015-07-23 Advanced Liquid Logic, Inc. Droplet actuator with improved waste disposal capability
GB201209924D0 (en) * 2012-06-05 2012-07-18 European Molecular Biology Lab Embl Microfluids for metagenomics
GB201115895D0 (en) 2011-09-14 2011-10-26 Embl Microfluidic device
EP2758173B1 (en) * 2011-09-19 2019-07-24 Centre National De La Recherche Scientifique Microfluidic system
CN103958050B (en) * 2011-09-28 2016-09-14 哈佛学院院长等 Systems and methods for droplet generation and/or fluid manipulation
EP2761191A4 (en) * 2011-09-30 2015-08-26 Univ California DEVICES AND METHODS FOR FLUID FLOW PROGRAMMING USING SEQUENCED MICROSTRUCTURES
US9896725B2 (en) * 2011-10-04 2018-02-20 The Regents Of The University Of California Corporation Real-time, label-free detection of macromolecules in droplets based on electrical measurements
US9228233B2 (en) 2011-10-17 2016-01-05 Good Start Genetics, Inc. Analysis methods
US10865440B2 (en) 2011-10-21 2020-12-15 IntegenX, Inc. Sample preparation, processing and analysis systems
US20150136604A1 (en) 2011-10-21 2015-05-21 Integenx Inc. Sample preparation, processing and analysis systems
WO2013078216A1 (en) 2011-11-21 2013-05-30 Advanced Liquid Logic Inc Glucose-6-phosphate dehydrogenase assays
US9970984B2 (en) 2011-12-01 2018-05-15 Life Technologies Corporation Method and apparatus for identifying defects in a chemical sensor array
US10222391B2 (en) 2011-12-07 2019-03-05 The Johns Hopkins University System and method for screening a library of samples
US8883088B2 (en) 2011-12-23 2014-11-11 California Institute Of Technology Sample preparation devices and systems
US9518291B2 (en) 2011-12-23 2016-12-13 California Institute Of Technology Devices and methods for biological sample-to-answer and analysis
WO2013101741A1 (en) 2011-12-30 2013-07-04 Abbott Molecular, Inc. Channels with cross-sectional thermal gradients
US8821798B2 (en) 2012-01-19 2014-09-02 Life Technologies Corporation Titanium nitride as sensing layer for microwell structure
US8747748B2 (en) 2012-01-19 2014-06-10 Life Technologies Corporation Chemical sensor with conductive cup-shaped sensor surface
IN2014DN06644A (en) * 2012-02-08 2015-05-22 Harvard College
EP2823303A4 (en) * 2012-02-10 2015-09-30 Raindance Technologies Inc MOLECULAR DIAGNOSTIC SCREEN TYPE ASSAY
AU2013220890B2 (en) 2012-02-16 2016-03-10 National Research Council Of Canada Centrifugal microfluidic mixing apparatus and method
WO2013126741A1 (en) 2012-02-24 2013-08-29 Raindance Technologies, Inc. Labeling and sample preparation for sequencing
WO2013143733A1 (en) * 2012-03-27 2013-10-03 Asml Netherlands B.V. Fuel system for lithographic apparatus, euv source,lithographic apparatus and fuel filtering method
US8209130B1 (en) 2012-04-04 2012-06-26 Good Start Genetics, Inc. Sequence assembly
US8812422B2 (en) 2012-04-09 2014-08-19 Good Start Genetics, Inc. Variant database
WO2013155531A2 (en) 2012-04-13 2013-10-17 Bio-Rad Laboratories, Inc. Sample holder with a well having a wicking promoter
US10227635B2 (en) 2012-04-16 2019-03-12 Molecular Loop Biosolutions, Llc Capture reactions
US9910012B2 (en) * 2012-04-19 2018-03-06 Wake Pure Chemical Industries, Ltd. Methods for real-time sampling of reaction products
AU2013249007B2 (en) 2012-04-20 2016-04-21 California Institute Of Technology Fluidic devices and systems for sample preparation or autonomous analysis
WO2013159116A1 (en) 2012-04-20 2013-10-24 University Of Chicago Fluidic devices for biospecimen preservation
US9803237B2 (en) 2012-04-24 2017-10-31 California Institute Of Technology Slip-induced compartmentalization
EP3524693A1 (en) 2012-04-30 2019-08-14 Raindance Technologies, Inc. Digital analyte analysis
US20130292247A1 (en) * 2012-05-01 2013-11-07 Commissariat A L'energie Atomique Et Aux Ene Alt Method and microsystem for the determination of clausius-mossotti factors for colloidal particles
US9429565B2 (en) 2012-05-08 2016-08-30 Cellecta, Inc. Clonal analysis of functional genomic assays and compositions for practicing same
US8786331B2 (en) 2012-05-29 2014-07-22 Life Technologies Corporation System for reducing noise in a chemical sensor array
WO2013192351A1 (en) 2012-06-20 2013-12-27 Bio-Rad Laboratories, Inc. Stabilized droplets for calibration and testing
TWI475214B (en) * 2012-06-22 2015-03-01 Wistron Corp Biological particle analyzer and method of analyzing biological particles
BR112014032727B1 (en) 2012-06-27 2021-12-14 Illumina France METHOD AND SYSTEM FOR PERFORMING DROP OPERATIONS ON A DROPOLE ON A DROP ACTUATOR TO REDUCE BUBBLE FORMATION
WO2014028378A2 (en) * 2012-08-13 2014-02-20 The Regents Of The University Of California Methods and systems for detecting biological components
US10584381B2 (en) 2012-08-14 2020-03-10 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10273541B2 (en) 2012-08-14 2019-04-30 10X Genomics, Inc. Methods and systems for processing polynucleotides
EP2885418A4 (en) * 2012-08-14 2016-03-02 10X Genomics Inc MICROCAPSULE COMPOSITIONS AND METHODS
US9951386B2 (en) 2014-06-26 2018-04-24 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10752949B2 (en) 2012-08-14 2020-08-25 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10221442B2 (en) 2012-08-14 2019-03-05 10X Genomics, Inc. Compositions and methods for sample processing
US9701998B2 (en) 2012-12-14 2017-07-11 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10323279B2 (en) 2012-08-14 2019-06-18 10X Genomics, Inc. Methods and systems for processing polynucleotides
US11591637B2 (en) 2012-08-14 2023-02-28 10X Genomics, Inc. Compositions and methods for sample processing
EP2703497B1 (en) * 2012-08-31 2016-06-22 Roche Diagniostics GmbH Microfluidic chip, device and system for the generation of aqueous droplets in emulsion oil for nucleic acid amplification
US9790546B2 (en) 2012-08-31 2017-10-17 Roche Molecular Systems, Inc. Microfluidic chip, device and system for the generation of aqueous droplets in emulsion oil for nucleic acid amplification
EP2895591A4 (en) * 2012-09-12 2016-10-12 Gnubio Inc INTEGRATED MICROFLUIDIC SYSTEM, METHOD AND KIT FOR PERFORMING TESTS
CA2922537C (en) * 2012-09-12 2022-03-22 The Regents Of The University Of California Accurate genome sequencing of single cells by single-stranded amplification and sequencing
US9981273B2 (en) 2012-09-28 2018-05-29 The Board Of Trustees Of The Leland Stanford Junior University Negative dielectrophoresis for selective elution of immuno-bound particles
US10000802B2 (en) 2012-10-04 2018-06-19 Base4 Innovation Ltd Sequencing method
GB201217772D0 (en) * 2012-10-04 2012-11-14 Base4 Innovation Ltd Sequencing method
US9284520B2 (en) 2012-11-05 2016-03-15 California Institute Of Technology Instruments for biological sample preparation devices
US8963095B2 (en) 2012-11-27 2015-02-24 Diagnostic Chips, LLC Electrokinetic microfluidic flow cytometer apparatuses with differential resistive particle counting and optical sorting
US10525465B2 (en) 2012-11-29 2020-01-07 Koninklijke Philips N.V. Cartridge for uptake and processing of a sample
EP2925447B1 (en) 2012-11-30 2020-04-08 The Broad Institute, Inc. High-throughput dynamic reagent delivery system
US10533221B2 (en) 2012-12-14 2020-01-14 10X Genomics, Inc. Methods and systems for processing polynucleotides
WO2014093676A1 (en) 2012-12-14 2014-06-19 10X Technologies, Inc. Methods and systems for processing polynucleotides
US9080968B2 (en) 2013-01-04 2015-07-14 Life Technologies Corporation Methods and systems for point of use removal of sacrificial material
US9841398B2 (en) 2013-01-08 2017-12-12 Life Technologies Corporation Methods for manufacturing well structures for low-noise chemical sensors
EP2948703B1 (en) 2013-01-25 2019-03-13 Bio-Rad Laboratories, Inc. System and method for performing droplet inflation
US9752181B2 (en) 2013-01-26 2017-09-05 Denovo Sciences, Inc. System and method for capturing and analyzing cells
US8962366B2 (en) 2013-01-28 2015-02-24 Life Technologies Corporation Self-aligned well structures for low-noise chemical sensors
WO2014121241A1 (en) 2013-02-01 2014-08-07 Bio-Rad Laboratories, Inc. System for detection of spaced droplets
CA2900481A1 (en) 2013-02-08 2014-08-14 10X Genomics, Inc. Polynucleotide barcode generation
GB201303507D0 (en) * 2013-02-27 2013-04-10 Hollfelder Florian Assays
US8963216B2 (en) 2013-03-13 2015-02-24 Life Technologies Corporation Chemical sensor with sidewall spacer sensor surface
US8841217B1 (en) 2013-03-13 2014-09-23 Life Technologies Corporation Chemical sensor with protruded sensor surface
US9707562B2 (en) 2013-03-13 2017-07-18 Denovo Sciences, Inc. System for capturing and analyzing cells
WO2014153071A1 (en) 2013-03-14 2014-09-25 The Broad Institute, Inc. Methods for quantitating dna using digital multiple displacement amplification
WO2014152421A1 (en) 2013-03-14 2014-09-25 Good Start Genetics, Inc. Methods for analyzing nucleic acids
WO2014149779A1 (en) 2013-03-15 2014-09-25 Life Technologies Corporation Chemical device with thin conductive element
US10119134B2 (en) 2013-03-15 2018-11-06 Abvitro Llc Single cell bar-coding for antibody discovery
US9835585B2 (en) 2013-03-15 2017-12-05 Life Technologies Corporation Chemical sensor with protruded sensor surface
CN105264366B (en) 2013-03-15 2019-04-16 生命科技公司 Chemical sensors with consistent sensor surface area
CN108445242B (en) 2013-03-15 2022-03-04 Hycor生物医学有限责任公司 Device for performing luminescence and fluorescence measurements of a sample and related method
US9116117B2 (en) 2013-03-15 2015-08-25 Life Technologies Corporation Chemical sensor with sidewall sensor surface
EP2972279B1 (en) 2013-03-15 2021-10-06 Life Technologies Corporation Chemical sensors with consistent sensor surface areas
JP6609544B2 (en) * 2013-03-15 2019-11-20 ラリアット・バイオサイエンシズ・インコーポレイテッド Microfluidic methods for handling DNA
CN110082550A (en) * 2013-03-16 2019-08-02 莱斯利·唐·罗伯茨 Self-contained modular cartridges and programmable reagent delivery systems
WO2014165559A2 (en) * 2013-04-02 2014-10-09 Raindance Technologies, Inc. Systems and methods for handling microfluidic droplets
GB201306444D0 (en) * 2013-04-09 2013-05-22 Base4 Innovation Ltd Single nucleotide detection method
WO2014172288A2 (en) 2013-04-19 2014-10-23 Raindance Technologies, Inc. Digital analyte analysis
US20140336063A1 (en) 2013-05-09 2014-11-13 Life Technologies Corporation Windowed Sequencing
CN105431575B (en) * 2013-05-09 2017-08-29 生物辐射实验室股份有限公司 Magnetic Immuno-Digital PCR Assay
US9809851B2 (en) 2013-05-29 2017-11-07 Bio-Rad Laboratories, Inc. Systems and methods for sequencing in emulsion based microfluidics
CA2948452C (en) * 2013-05-31 2021-04-13 Rajesh Krishnamurthy Rapid microbial detection
US9856535B2 (en) 2013-05-31 2018-01-02 Denovo Sciences, Inc. System for isolating cells
US10391490B2 (en) 2013-05-31 2019-08-27 Celsee Diagnostics, Inc. System and method for isolating and analyzing cells
WO2014197377A2 (en) 2013-06-03 2014-12-11 Good Start Genetics, Inc. Methods and systems for storing sequence read data
US10458942B2 (en) 2013-06-10 2019-10-29 Life Technologies Corporation Chemical sensor array having multiple sensors per well
WO2014210207A1 (en) 2013-06-25 2014-12-31 University Of Washington Through Its Center For Commercialization Self-digitization of sample volumes
GB201311679D0 (en) 2013-06-28 2013-08-14 Ibm Microfluidic chip with dielectrophoretic electrodes extending in hydrophilic flow path
CN110988373A (en) 2013-07-05 2020-04-10 华盛顿大学商业中心 Methods, compositions and systems for microfluidic analysis
US8961904B2 (en) 2013-07-16 2015-02-24 Premium Genetics (Uk) Ltd. Microfluidic chip
CN105555972B (en) 2013-07-25 2020-07-31 伯乐生命医学产品有限公司 Genetic assay
GB201313583D0 (en) 2013-07-30 2013-09-11 Sphere Fluidics Ltd Emulsion
GB2516684A (en) 2013-07-30 2015-02-04 Sphere Fluidics Ltd Microfluidic devices and systems
EP3039119A4 (en) * 2013-08-27 2017-04-05 GnuBIO, Inc. Microfluidic devices and methods of their use
US10395758B2 (en) 2013-08-30 2019-08-27 10X Genomics, Inc. Sequencing methods
DE102013217959A1 (en) 2013-09-09 2015-03-12 Efficient Robotics Gmbh Microfluidic analysis device and manufacturing process
US20160231324A1 (en) * 2013-09-24 2016-08-11 The Regents Of The University Of California Encapsulated sensors and sensing systems for bioassays and diagnostics and methods for making and using them
WO2015048798A1 (en) 2013-09-30 2015-04-02 Gnubio, Inc. Microfluidic cartridge device and methods of use and assembly
US11901041B2 (en) 2013-10-04 2024-02-13 Bio-Rad Laboratories, Inc. Digital analysis of nucleic acid modification
FR3011932B1 (en) * 2013-10-10 2017-03-24 Univ Strasbourg TIME-RESOLVED FLUORESCENCE MEASUREMENT DEVICE AND METHOD FOR HIGH-SPEED SAMPLE SCREENING
US10851414B2 (en) 2013-10-18 2020-12-01 Good Start Genetics, Inc. Methods for determining carrier status
US11041203B2 (en) 2013-10-18 2021-06-22 Molecular Loop Biosolutions, Inc. Methods for assessing a genomic region of a subject
JP6385356B2 (en) * 2013-10-25 2018-09-05 一般財団法人生産技術研究奨励会 DNA detection method
US11796449B2 (en) 2013-10-30 2023-10-24 Abs Global, Inc. Microfluidic system and method with focused energy apparatus
WO2015069634A1 (en) 2013-11-08 2015-05-14 President And Fellows Of Harvard College Microparticles, methods for their preparation and use
US20150159224A1 (en) * 2013-11-13 2015-06-11 Huidan ZHANG Ultrasensitive detection and characterization of clustered kras mutations using peptide nucleic acid clamp pcr in drop-based microfluidics
US20150138337A1 (en) * 2013-11-15 2015-05-21 Schlumberger Technology Corporation Imaging-Based Measurement Device
WO2015074004A1 (en) 2013-11-17 2015-05-21 Quantum-Si Incorporated Integrated device with external light source for probing detecting and analyzing molecules
US10191071B2 (en) 2013-11-18 2019-01-29 IntegenX, Inc. Cartridges and instruments for sample analysis
WO2015074898A1 (en) * 2013-11-19 2015-05-28 Qiagen Gmbh Method for generating emulsions
WO2015077717A1 (en) 2013-11-25 2015-05-28 The Broad Institute Inc. Compositions and methods for diagnosing, evaluating and treating cancer by means of the dna methylation status
EP3074122A4 (en) 2013-11-27 2017-11-29 Bio-Rad Laboratories, Inc. Microfluidic droplet packing
WO2015085147A1 (en) 2013-12-05 2015-06-11 The Broad Institute Inc. Polymorphic gene typing and somatic change detection using sequencing data
US9944977B2 (en) 2013-12-12 2018-04-17 Raindance Technologies, Inc. Distinguishing rare variations in a nucleic acid sequence from a sample
US9824068B2 (en) 2013-12-16 2017-11-21 10X Genomics, Inc. Methods and apparatus for sorting data
JP7060324B2 (en) 2013-12-20 2022-04-26 ザ・ブロード・インスティテュート・インコーポレイテッド Combination therapy with neoantigen vaccine
EP3090063B1 (en) 2013-12-31 2019-11-06 Bio-Rad Laboratories, Inc. Method for detection of latent retrovirus
TWI526392B (en) 2014-01-21 2016-03-21 國立清華大學 Method for forming microfluidic structure
CA2937784C (en) 2014-01-24 2022-08-23 The Johns Hopkins University System and device for high throughput generation of combinatorial droplets and methods of use
EP3584306B1 (en) * 2014-01-31 2025-07-02 Toppan Printing Co., Ltd. Biomolecule analysis kit and biomolecule analysis method
JP2017508457A (en) 2014-02-27 2017-03-30 ザ・ブロード・インスティテュート・インコーポレイテッド T cell balance gene expression, composition and method of use thereof
US9726653B2 (en) 2014-03-26 2017-08-08 Hamilton Sundstrand Corporation Chemical detector
US9694361B2 (en) 2014-04-10 2017-07-04 10X Genomics, Inc. Fluidic devices, systems, and methods for encapsulating and partitioning reagents, and applications of same
US11053548B2 (en) 2014-05-12 2021-07-06 Good Start Genetics, Inc. Methods for detecting aneuploidy
EP3142790B8 (en) 2014-05-14 2019-04-24 Hooke Bio Limited Method for handling fluid in a microfluidic device with channel plates
GB2544198B (en) 2014-05-21 2021-01-13 Integenx Inc Fluidic cartridge with valve mechanism
WO2015191723A1 (en) * 2014-06-10 2015-12-17 University Of Southern California Usc Discrete elements for 3d microfluidics
CA2950043C (en) 2014-06-11 2022-08-02 Samplix S.A.R.L. Nucleotide sequence exclusion enrichment by droplet sorting (needls)
CN106536709A (en) 2014-06-16 2017-03-22 基纽拜奥股份有限公司 Size alternating injection into drops to facilitate sorting
WO2015196065A1 (en) * 2014-06-19 2015-12-23 The Trustees Of The University Of Pennsylvania Apparatus and methods for making recombinant protein-stabilized monodisperse microbubbles
US10839939B2 (en) 2014-06-26 2020-11-17 10X Genomics, Inc. Processes and systems for nucleic acid sequence assembly
EP3161160B1 (en) 2014-06-26 2021-10-13 10X Genomics, Inc. Methods of analyzing nucleic acids from individual cells or cell populations
US12312640B2 (en) 2014-06-26 2025-05-27 10X Genomics, Inc. Analysis of nucleic acid sequences
CN106536756A (en) 2014-06-26 2017-03-22 10X基因组学有限公司 Analysis of nucleic acid sequences
US10697007B2 (en) 2014-06-27 2020-06-30 The Regents Of The University Of California PCR-activated sorting (PAS)
CN106573243B (en) * 2014-08-04 2019-07-05 基因细胞生物系统有限公司 Three Phase Fluid Handling
GB201414177D0 (en) * 2014-08-11 2014-09-24 Sphere Fluidics Ltd Droplet Sorting
US20160048608A1 (en) 2014-08-15 2016-02-18 Good Start Genetics, Inc. Systems and methods for genetic analysis
WO2016028378A1 (en) * 2014-08-21 2016-02-25 Schlumberger Canada Limited Measurement of liquid parameters using a microfluidic device
WO2016040476A1 (en) 2014-09-09 2016-03-17 The Broad Institute, Inc. A droplet-based method and apparatus for composite single-cell nucleic acid analysis
WO2016040446A1 (en) 2014-09-10 2016-03-17 Good Start Genetics, Inc. Methods for selectively suppressing non-target sequences
US10590483B2 (en) 2014-09-15 2020-03-17 Abvitro Llc High-throughput nucleotide library sequencing
US10429399B2 (en) 2014-09-24 2019-10-01 Good Start Genetics, Inc. Process control for increased robustness of genetic assays
SG11201702557XA (en) * 2014-09-29 2017-04-27 Chipcare Corp A device for optical detection of cells comprising a cartridge and fluidic chip and methods thereof
CN106715672A (en) * 2014-09-30 2017-05-24 国立研究开发法人科学技术振兴机构 Bubble jetting chip, local ablation device and local ablation method, and injection device and injection method
US10210410B2 (en) 2014-10-22 2019-02-19 Integenx Inc. Systems and methods for biometric data collections
EP3209419B1 (en) 2014-10-22 2024-12-11 The Regents of The University of California High definition microdroplet printer
CN113092563B (en) 2014-10-22 2024-06-07 尹特根埃克斯有限公司 Systems and methods for sample preparation, processing, and analysis
WO2016065365A1 (en) * 2014-10-24 2016-04-28 Sandia Corporation Method and device for tracking and manipulation of droplets
CA2964472A1 (en) 2014-10-29 2016-05-06 10X Genomics, Inc. Methods and compositions for targeted nucleic acid sequencing
US10000799B2 (en) 2014-11-04 2018-06-19 Boreal Genomics, Inc. Methods of sequencing with linked fragments
US9975122B2 (en) 2014-11-05 2018-05-22 10X Genomics, Inc. Instrument systems for integrated sample processing
EP3017868A1 (en) * 2014-11-10 2016-05-11 Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie - Hans-Knöll-Institut - Device and method for extracting individual picoliter droplets from microfluidic emulsions for further analysis and scale-up
JP6742311B2 (en) 2014-11-14 2020-08-19 アテナ ダイアグナスティクス，インコーポレイテッド Method for detecting silent carrier genotype
US9636675B2 (en) 2014-11-26 2017-05-02 International Business Machines Corporation Pillar array structure with uniform and high aspect ratio nanometer gaps
US11478789B2 (en) * 2014-11-26 2022-10-25 Medica Corporation Automated microscopic cell analysis
US10625259B1 (en) 2014-11-26 2020-04-21 Medica Corporation Automated microscopic cell analysis
US12005441B1 (en) * 2014-11-26 2024-06-11 Medica Corporation Automated microscopic cell analysis
US20170328924A1 (en) 2014-11-26 2017-11-16 Ronald Jones Automated microscopic cell analysis
EP3224594A4 (en) 2014-11-28 2018-08-08 Chipcare Corporation Multiplex bead array assay
US10077472B2 (en) 2014-12-18 2018-09-18 Life Technologies Corporation High data rate integrated circuit with power management
KR20170097712A (en) 2014-12-18 2017-08-28 라이프 테크놀로지스 코포레이션 Methods and apparatus for measuring analytes using large scale fet arrays
CN107250784B (en) 2014-12-18 2020-10-23 生命科技公司 High data rate integrated circuit with transmitter configuration
EP3234193B1 (en) 2014-12-19 2020-07-15 Massachusetts Institute of Technology Molecular biomarkers for cancer immunotherapy
ES3049406T3 (en) 2014-12-19 2025-12-16 Broad Inst Inc Methods for profiling the t-cell-receptor repertoire
US10066259B2 (en) 2015-01-06 2018-09-04 Good Start Genetics, Inc. Screening for structural variants
CN107427808B (en) 2015-01-12 2020-10-23 10X基因组学有限公司 Method and system for preparing nucleic acid sequencing library and library prepared by using same
KR20170106979A (en) 2015-01-13 2017-09-22 10엑스 제노믹스, 인크. System and method for visualizing structure variation and phase adjustment information
WO2016126871A2 (en) 2015-02-04 2016-08-11 The Regents Of The University Of California Sequencing of nucleic acids via barcoding in discrete entities
KR20240116531A (en) 2015-02-09 2024-07-29 슬링샷 바이오사이언시즈 인코포레이티드 Hydrogel particles with tunable optical properties and methods for using the same
MX2017010142A (en) 2015-02-09 2017-12-11 10X Genomics Inc Systems and methods for determining structural variation and phasing using variant call data.
BR112017018054A2 (en) 2015-02-24 2018-07-24 10X Genomics Inc Methods for Covering Targeted Nucleic Acid Sequences
WO2016137973A1 (en) 2015-02-24 2016-09-01 10X Genomics Inc Partition processing methods and systems
WO2016138488A2 (en) 2015-02-26 2016-09-01 The Broad Institute Inc. T cell balance gene expression, compositions of matters and methods of use thereof
CN107615038B (en) * 2015-03-06 2021-01-29 美铝澳大利亚有限公司 Online sampling device
CN107614700A (en) 2015-03-11 2018-01-19 布罗德研究所有限公司 Genotype and phenotype coupling
WO2016149661A1 (en) * 2015-03-18 2016-09-22 The Broad Institute, Inc. Massively parallel on-chip coalescence of microemulsions
EP3271477B1 (en) * 2015-03-20 2020-06-24 Novozymes A/S Droplet-based selection by injection
US20180071981A1 (en) 2015-03-31 2018-03-15 The Regents Of The University Of California System and method for tunable patterning and assembly of particles via acoustophoresis
CA3176115A1 (en) 2015-04-22 2016-10-27 Berkeley Lights, Inc. Microfluidic device for culturing biological cells and methods of use thereof
CR20230191A (en) 2015-05-20 2023-07-06 Dana Farber Cancer Inst Inc SHARED NEOANTIGENS (Div. exp 2017-584)
JP6713730B2 (en) * 2015-05-20 2020-06-24 シスメックス株式会社 Cell detection device and cell detection method
CN107405633A (en) 2015-05-22 2017-11-28 香港科技大学 Drop generator for inducing generation of drops based on high aspect ratio
GB201509640D0 (en) 2015-06-03 2015-07-15 Sphere Fluidics Ltd Systems and methods
WO2016205728A1 (en) 2015-06-17 2016-12-22 Massachusetts Institute Of Technology Crispr mediated recording of cellular events
EP3314046B1 (en) 2015-06-25 2019-04-24 Hooke Bio Limited Mechanical device for generating combinatorial library
WO2017004250A1 (en) 2015-06-29 2017-01-05 Arizona Board Of Regents, A Body Corporate Of The State Of Arizona, Acting For And On Behalf Of Arizona State University Systems and methods for continuous flow digital droplet polymerase chain reaction bioanalysis
JP6527406B2 (en) * 2015-07-01 2019-06-05 本田技研工業株式会社 Analytical battery and method of manufacturing the same
CN108027382B (en) 2015-07-07 2021-12-24 华盛顿大学 Systems, methods, and apparatus for sample self-digitization
US10976232B2 (en) 2015-08-24 2021-04-13 Gpb Scientific, Inc. Methods and devices for multi-step cell purification and concentration
US10647981B1 (en) 2015-09-08 2020-05-12 Bio-Rad Laboratories, Inc. Nucleic acid library generation methods and compositions
GB201516447D0 (en) 2015-09-16 2015-10-28 Sphere Fluidics Ltd Microfluidic structure
IL258248B (en) 2015-09-24 2022-07-01 Abvitro Llc Affinity-oligonucleotide conjugates and uses thereof
ES2978717T3 (en) 2015-09-24 2024-09-18 Abvitro Llc Single amplicon-triggered exclusion PCR
US10928392B2 (en) 2015-09-25 2021-02-23 Abvitro Llc High throughput process for T cell receptor target identification of natively-paired T cell receptor sequences
WO2017061620A1 (en) * 2015-10-09 2017-04-13 シスメックス株式会社 Analyte treatment chip, analyte treatment device, and analyte treatment method
CN108139418B (en) 2015-10-09 2022-06-07 希森美康株式会社 Subject processing chip, subject processing apparatus, and subject processing method
AU2016338907B2 (en) 2015-10-13 2022-07-07 President And Fellows Of Harvard College Systems and methods for making and using gel microspheres
US10799865B2 (en) 2015-10-27 2020-10-13 Berkeley Lights, Inc. Microfluidic apparatus having an optimized electrowetting surface and related systems and methods
EP3370868B1 (en) 2015-10-27 2020-12-09 Berkeley Lights, Inc. Microfluidic electrowetting device apparatus having a covalently bound hydrophobic surface
WO2017075294A1 (en) 2015-10-28 2017-05-04 The Board Institute Inc. Assays for massively combinatorial perturbation profiling and cellular circuit reconstruction
US11092607B2 (en) 2015-10-28 2021-08-17 The Board Institute, Inc. Multiplex analysis of single cell constituents
WO2017075297A1 (en) 2015-10-28 2017-05-04 The Broad Institute Inc. High-throughput dynamic reagent delivery system
US20190040461A1 (en) * 2015-11-04 2019-02-07 Celemics, Inc. Method for extracting and characterizing molecular clones
CN108463708B (en) 2015-11-06 2021-10-08 密歇根大学董事会 Droplet-based microfluidic rheometer system
WO2017087505A1 (en) * 2015-11-17 2017-05-26 The Regents Of The University Of Michigan Macromolecular structures and uses thereof
US11371094B2 (en) 2015-11-19 2022-06-28 10X Genomics, Inc. Systems and methods for nucleic acid processing using degenerate nucleotides
US20180345285A1 (en) * 2015-11-25 2018-12-06 Spectradyne Llc Systems and devices for microfluidic cartridge
JP6703824B2 (en) * 2015-11-30 2020-06-03 シスメックス株式会社 Cell selection method, cell detection method, cell selection device, and cell detection device
PT3882357T (en) 2015-12-04 2022-09-05 10X Genomics Inc Methods and compositions for nucleic acid analysis
US20170157605A1 (en) * 2015-12-04 2017-06-08 Justin Lock Methods and compositions for low volume liquid handling
US11009464B2 (en) * 2015-12-11 2021-05-18 International Business Machines Corporation Smartphone compatible on-chip biodetection using integrated optical component and microfluidic channel with nanopillar array
WO2017117567A1 (en) 2015-12-30 2017-07-06 Berkeley Lights, Inc. Droplet generation in a microfluidic device having an optoelectrowetting configuration
WO2017124101A2 (en) 2016-01-15 2017-07-20 The Broad Institute Inc. Semi-permeable arrays for analyzing biological systems and methods of using same
WO2017136751A1 (en) 2016-02-05 2017-08-10 The Broad Institute Inc. Multi-stage, multiplexed target isolation and processing from heterogeneous populations
JP6735348B2 (en) 2016-02-11 2020-08-05 １０エックス ジェノミクス， インコーポレイテッド Systems, methods and media for de novo assembly of whole genome sequence data
WO2017147196A1 (en) 2016-02-22 2017-08-31 Massachusetts Institute Of Technology Methods for identifying and modulating immune phenotypes
EP3443327B1 (en) * 2016-03-17 2023-06-07 Kazemzadeh, Amin Microfluidic on-chip storage devices
WO2017161325A1 (en) 2016-03-17 2017-09-21 Massachusetts Institute Of Technology Methods for identifying and modulating co-occurant cellular phenotypes
US12060412B2 (en) 2016-03-21 2024-08-13 The Broad Institute, Inc. Methods for determining spatial and temporal gene expression dynamics in single cells
GB201604876D0 (en) * 2016-03-22 2016-05-04 Uni I Tromsø Norges Arktiske Uni Marine DNA polymerases
ES2952832T3 (en) 2016-03-28 2023-11-06 Ncan Genomics Inc Linked Duplex Target Capture
US10961573B2 (en) 2016-03-28 2021-03-30 Boreal Genomics, Inc. Linked duplex target capture
ES2786974T3 (en) * 2016-04-07 2020-10-14 Illumina Inc Methods and systems for the construction of standard nucleic acid libraries
FR3050269B1 (en) * 2016-04-15 2018-05-11 Ecole Superieure De Physique Et De Chimie Industrielles De La Ville De Paris (Espci) METHOD FOR SELECTING AND RECOVERING PRODUCTS AND ASSOCIATED SYSTEM
FR3050212B1 (en) 2016-04-15 2020-09-25 Chu Montpellier TUMOR CELL DETECTION AND / OR CHARACTERIZATION METHOD AND ASSOCIATED APPARATUS
WO2017192286A1 (en) 2016-05-02 2017-11-09 Purdue Research Foundation Systems and methods for producing a chemical product
WO2017197343A2 (en) 2016-05-12 2017-11-16 10X Genomics, Inc. Microfluidic on-chip filters
WO2017197338A1 (en) 2016-05-13 2017-11-16 10X Genomics, Inc. Microfluidic systems and methods of use
JP7046415B2 (en) 2016-05-26 2022-04-04 バークレー ライツ，インコーポレイテッド Covalently modified surfaces, kits and methods of preparation and use
CN109689887A (en) 2016-07-08 2019-04-26 加州理工学院 For carrying out the method and apparatus for flowing through capture of analytes in low concentration
EP3481540B1 (en) 2016-07-08 2022-12-21 President and Fellows of Harvard College Formation of colloids or gels within droplets
US10124331B2 (en) * 2016-07-15 2018-11-13 Board Of Regents, The University Of Texas System Optofluidic lasers with surface gain and methods of making and using the same
US11339434B2 (en) * 2016-07-29 2022-05-24 The Regents Of The University Of California Methods for determining gene functions
WO2018031304A1 (en) * 2016-08-08 2018-02-15 Tru Tag Technologies, Inc. Identification of a tagged liquid
CN110088290A (en) 2016-08-10 2019-08-02 加利福尼亚大学董事会 Multiple displacement amplification and PCR are combined in lotion droplet
KR101816288B1 (en) * 2016-08-11 2018-01-09 한국기초과학지원연구원 Method of pre-treating sample with super absorbent polymer and device therefor
MX2019002754A (en) * 2016-09-12 2019-08-29 Abs Global Inc Method and system for hydrophobic coating of microfluidic chips.
US11872559B2 (en) 2016-09-14 2024-01-16 Ecole Polytechnique Federale De Lausanne (Epfl) Device for high throughput single-cell studies
US20180073062A1 (en) * 2016-09-15 2018-03-15 The University Of Chicago Compositions and methods for identifying endogenous dna-dna interactions
WO2018054975A1 (en) * 2016-09-20 2018-03-29 The European Molecular Biology Laboratory Microfluidic sorting devices and methods
JP6929354B2 (en) 2016-09-24 2021-09-01 アブビトロ， エルエルシー Affinity-oligonucleotide conjugates and their use
WO2018067965A1 (en) * 2016-10-07 2018-04-12 The Regents Of The University Of California Device and method for microscale chemical reactions
US11857967B2 (en) 2016-10-07 2024-01-02 University Of Maryland, College Park Integration of porous monolithic structures within microfluidic systems
FR3057356B1 (en) * 2016-10-11 2021-12-17 Commissariat Energie Atomique METHOD AND SYSTEM FOR CHARACTERIZING A FLUID
EP3315963A1 (en) * 2016-10-26 2018-05-02 Fuchs Petrolub SE Sample receiving element, analyses set and method for analyzing a liquid, in particular a cooling lubricant emulsion
CN110249050B (en) * 2016-11-08 2023-08-18 南纳治疗有限公司 Library of non-tag encoded compounds
EP3544737B1 (en) 2016-11-28 2025-04-23 Arizona Board of Regents on behalf of Arizona State University Methods related to continuous flow droplet reaction
JP6884562B2 (en) * 2016-11-30 2021-06-09 シスメックス株式会社 Specimen processing method and sample processing equipment
CA3045457A1 (en) * 2016-12-01 2018-06-07 Novel Microdevices, Llc Automated point-of-care devices for complex sample processing and methods of use thereof
JP7048609B2 (en) 2016-12-09 2022-04-05 ボリアル ジェノミクス， インコーポレイテッド Linked ligation
EP3571308A4 (en) 2016-12-21 2020-08-19 The Regents of The University of California SINGLE CELL GENOMIC SEQUENCING WITH DROPLETS BASED ON HYDROGEL
US10815525B2 (en) 2016-12-22 2020-10-27 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10550429B2 (en) 2016-12-22 2020-02-04 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10011872B1 (en) 2016-12-22 2018-07-03 10X Genomics, Inc. Methods and systems for processing polynucleotides
EP3562893B1 (en) * 2016-12-28 2024-06-19 Bio-Rad Laboratories, Inc. Modification of surface properties of microfluidic devices
DK3351302T3 (en) 2017-01-18 2021-08-09 Biomillenia Sas Microfluidic system and method with carefully controlled incubation time and controlled conditions
EP3574116A1 (en) 2017-01-24 2019-12-04 The Broad Institute, Inc. Compositions and methods for detecting a mutant variant of a polynucleotide
CN117512066A (en) 2017-01-30 2024-02-06 10X基因组学有限公司 Methods and systems for droplet-based single cell barcoding
US12264411B2 (en) 2017-01-30 2025-04-01 10X Genomics, Inc. Methods and systems for analysis
US10995333B2 (en) 2017-02-06 2021-05-04 10X Genomics, Inc. Systems and methods for nucleic acid preparation
JP7071056B2 (en) 2017-02-27 2022-05-18 シスメックス株式会社 Liquid delivery method and liquid delivery device
DE102017105194A1 (en) * 2017-03-10 2018-09-13 Little Things Factory Gmbh Focusing device, drop generator and method for generating a plurality of droplets
US12180546B2 (en) 2017-03-17 2024-12-31 Massachusetts Institute Of Technology Methods for identifying and modulating co-occurant cellular phenotypes
WO2018183744A1 (en) 2017-03-29 2018-10-04 The Research Foundation For The State University Of New York Microfluidic device and methods
US10508976B1 (en) 2017-03-31 2019-12-17 Advanced Micro Instruments, Inc. Gas sampling device and method
KR20190131572A (en) * 2017-04-05 2019-11-26 인디안 인스티튜트 오브 테크놀러지 마드라스(아이아이티 마드라스) Microfax for detection and isolation of target cells
JP6937382B2 (en) * 2017-04-14 2021-09-22 アドバンスト・ソリューションズ・ライフ・サイエンシズ，エルエルシー Column array and how to create a column array
EP3612629A1 (en) 2017-04-18 2020-02-26 The Broad Institute, Inc. Compositions for detecting secretion and methods of use
CN114643087A (en) * 2017-04-26 2022-06-21 伯克利之光生命科技公司 Biological treatment system and method using microfluidic devices with optimized electrowetting surfaces
US12534750B2 (en) 2017-05-02 2026-01-27 The University Of Tokyo Method for monitoring dynamic changes in cells or substance derived therefrom, and method for classifying cell using same
US11072816B2 (en) 2017-05-03 2021-07-27 The Broad Institute, Inc. Single-cell proteomic assay using aptamers
WO2018208447A1 (en) * 2017-05-11 2018-11-15 Emd Millipore Corporation Mechanical method of maintaining narrow residence time distributions in continuous flow systems
WO2018208448A1 (en) 2017-05-11 2018-11-15 Emd Millipore Corporation Method of maintaining narrow residence time distributions in continuous flow systems
CN119570912A (en) 2017-05-15 2025-03-07 贝斯4创新公司 Single nucleotide detection methods and related probes
US10544413B2 (en) * 2017-05-18 2020-01-28 10X Genomics, Inc. Methods and systems for sorting droplets and beads
EP4215616B1 (en) * 2017-05-18 2024-09-04 10X Genomics, Inc. Methods and systems for sorting droplets and beads
EP3625715A4 (en) 2017-05-19 2021-03-17 10X Genomics, Inc. SYSTEMS AND PROCEDURES FOR THE ANALYSIS OF DATA SETS
US20180340169A1 (en) 2017-05-26 2018-11-29 10X Genomics, Inc. Single cell analysis of transposase accessible chromatin
EP3445876B1 (en) 2017-05-26 2023-07-05 10X Genomics, Inc. Single cell analysis of transposase accessible chromatin
EP3631012B1 (en) 2017-05-26 2022-06-08 AbVitro LLC High-throughput polynucleotide library sequencing and transcriptome analysis
US10639607B2 (en) * 2017-06-16 2020-05-05 Matralix Pte Ltd Systems and methods for preparing wax and lipid particles
GB2566002B (en) 2017-06-22 2019-10-23 Sphere Fluidics Ltd Droplet dispensing systems
EP3645167A4 (en) 2017-06-28 2021-02-24 Bio-rad Laboratories, Inc. SYSTEM AND METHOD FOR DROPLET DETECTION
US10545516B2 (en) * 2017-08-02 2020-01-28 Schneider Electric Systems Usa, Inc. Industrial process control transmitter for determining solution concentration
WO2019040599A1 (en) * 2017-08-22 2019-02-28 Elegen Corporation Positional tracking and encoding in microfluidic devices
US10610865B2 (en) 2017-08-22 2020-04-07 10X Genomics, Inc. Droplet forming devices and system with differential surface properties
WO2019046307A1 (en) 2017-08-29 2019-03-07 Celsee Diagnostics, Inc. System and method for isolating and analyzing cells
EP3679370A1 (en) 2017-09-07 2020-07-15 Juno Therapeutics, Inc. Methods of identifying cellular attributes related to outcomes associated with cell therapy
US10837047B2 (en) 2017-10-04 2020-11-17 10X Genomics, Inc. Compositions, methods, and systems for bead formation using improved polymers
US10501739B2 (en) 2017-10-18 2019-12-10 Mission Bio, Inc. Method, systems and apparatus for single cell analysis
GB201717103D0 (en) * 2017-10-18 2017-11-29 Tech Partnership Plc Fluid ejector system
US11732257B2 (en) 2017-10-23 2023-08-22 Massachusetts Institute Of Technology Single cell sequencing libraries of genomic transcript regions of interest in proximity to barcodes, and genotyping of said libraries
WO2019084043A1 (en) 2017-10-26 2019-05-02 10X Genomics, Inc. Methods and systems for nuclecic acid preparation and chromatin analysis
WO2019083852A1 (en) 2017-10-26 2019-05-02 10X Genomics, Inc. Microfluidic channel networks for partitioning
EP3700672B1 (en) 2017-10-27 2022-12-28 10X Genomics, Inc. Methods for sample preparation and analysis
US11045805B2 (en) 2017-11-01 2021-06-29 Bio-Rad Laboratories, Inc. Microfluidic system and method for arranging objects
US20200407571A1 (en) * 2017-11-01 2020-12-31 University Of Connecticut Two-Dimensional Sheet Stabilized Emulsion Based Inks
JP7030361B2 (en) * 2017-11-06 2022-03-07 北京新▲い▼生物科技有限公司 Microdroplet generator
CN109746058B (en) * 2017-11-06 2024-05-24 北京新羿生物科技有限公司 Micro droplet detection chip
US12221720B2 (en) 2017-11-13 2025-02-11 The Broad Institute, Inc. Methods for determining spatial and temporal gene expression dynamics during adult neurogenesis in single cells
US11047845B1 (en) 2017-11-15 2021-06-29 Medica Corporation Control material and methods for cell analyzers
EP3625361A1 (en) 2017-11-15 2020-03-25 10X Genomics, Inc. Functionalized gel beads
US10829815B2 (en) 2017-11-17 2020-11-10 10X Genomics, Inc. Methods and systems for associating physical and genetic properties of biological particles
WO2019108851A1 (en) 2017-11-30 2019-06-06 10X Genomics, Inc. Systems and methods for nucleic acid preparation and analysis
WO2019110591A1 (en) * 2017-12-06 2019-06-13 Samplix Aps A microfluidic device and a method for provision of emulsion droplets
WO2019113506A1 (en) 2017-12-07 2019-06-13 The Broad Institute, Inc. Methods and compositions for multiplexing single cell and single nuclei sequencing
CN111699388B (en) 2017-12-12 2024-08-02 10X基因组学有限公司 Systems and methods for single cell processing
WO2019126789A1 (en) 2017-12-22 2019-06-27 10X Genomics, Inc. Systems and methods for processing nucleic acid molecules from one or more cells
EP3752832A1 (en) 2018-02-12 2020-12-23 10X Genomics, Inc. Methods characterizing multiple analytes from individual cells or cell populations
US11639928B2 (en) 2018-02-22 2023-05-02 10X Genomics, Inc. Methods and systems for characterizing analytes from individual cells or cell populations
WO2019169028A1 (en) 2018-02-28 2019-09-06 10X Genomics, Inc. Transcriptome sequencing through random ligation
WO2019169347A1 (en) 2018-03-02 2019-09-06 10X Genomics, Inc. Systems and apparatus for holding plates
US11841371B2 (en) 2018-03-13 2023-12-12 The Broad Institute, Inc. Proteomics and spatial patterning using antenna networks
EP3543351B1 (en) * 2018-03-19 2022-08-10 Ricoh Company, Ltd. Nucleic acid sample-contained container, method for producing nucleic acid sample-contained container, and nucleic acid sample
US11471884B2 (en) 2018-04-02 2022-10-18 Dropworks, Inc. Systems and methods for serial flow emulsion processes
US11485944B2 (en) 2018-04-04 2022-11-01 Hewlett-Packard Development Company, L.P. Microfluidic cellular membrane modification devices
CN112262218B (en) 2018-04-06 2024-11-08 10X基因组学有限公司 Systems and methods for quality control in single cell processing
WO2019217758A1 (en) 2018-05-10 2019-11-14 10X Genomics, Inc. Methods and systems for molecular library generation
WO2019226790A1 (en) 2018-05-23 2019-11-28 Abs Global, Inc. Systems and methods for particle focusing in microchannels
US11932899B2 (en) 2018-06-07 2024-03-19 10X Genomics, Inc. Methods and systems for characterizing nucleic acid molecules
US11703427B2 (en) 2018-06-25 2023-07-18 10X Genomics, Inc. Methods and systems for cell and bead processing
US12188014B1 (en) 2018-07-25 2025-01-07 10X Genomics, Inc. Compositions and methods for nucleic acid processing using blocking agents
US20200032335A1 (en) 2018-07-27 2020-01-30 10X Genomics, Inc. Systems and methods for metabolome analysis
EP3830573B1 (en) * 2018-07-29 2024-10-16 KOC Universitesi Microfluidic thromboelastometry instrument
KR102083845B1 (en) * 2018-07-31 2020-03-03 광주과학기술원 Blood diagnostic element
US11618025B2 (en) * 2018-08-01 2023-04-04 University Of Macau Apparatus and method for on-chip microfluids dispensing
US12163179B2 (en) 2018-08-03 2024-12-10 10X Gemomics, Inc. Methods and systems to minimize barcode exchange
CA3107376A1 (en) 2018-08-08 2020-02-13 Inivata Ltd. Method of sequencing using variable replicate multiplex pcr
CN110819702B (en) * 2018-08-13 2023-03-21 上海新微技术研发中心有限公司 Digital PCR system and digital PCR liquid drop forming method
EP3837377A4 (en) * 2018-08-17 2022-05-18 The Regents of University of California MONODISPERSED PARTICULATE INTRODUCED DROPLET FORMATION FROM STABLE JETS
US12065688B2 (en) 2018-08-20 2024-08-20 10X Genomics, Inc. Compositions and methods for cellular processing
WO2020041148A1 (en) 2018-08-20 2020-02-27 10X Genomics, Inc. Methods and systems for detection of protein-dna interactions using proximity ligation
WO2020039261A1 (en) 2018-08-23 2020-02-27 Boreal Genomics, Inc. Linked target capture and ligation
JP2020031606A (en) * 2018-08-31 2020-03-05 シスメックス株式会社 Nucleic acid detection device and nucleic acid detection method
CN110964628A (en) * 2018-09-29 2020-04-07 青岛华大智造普惠科技有限公司 Micro-fluidic chip system and preparation method of single-cell micro-droplets
US11130120B2 (en) * 2018-10-01 2021-09-28 Lifeng XIAO Micro-pipette tip for forming micro-droplets
WO2020077236A1 (en) 2018-10-12 2020-04-16 The Broad Institute, Inc. Method for extracting nuclei or whole cells from formalin-fixed paraffin-embedded tissues
GB201817321D0 (en) 2018-10-24 2018-12-05 Nanna Therapeutics Ltd Microbeads for tagless encoded chemical library screening
US11512356B2 (en) * 2018-11-08 2022-11-29 Tokitae Llc Systems and methods for particle multiplexing in droplets
US12165743B2 (en) 2018-11-09 2024-12-10 The Broad Institute, Inc. Compressed sensing for screening and tissue imaging
KR102089342B1 (en) * 2018-11-13 2020-04-20 (주)아프로텍 Precipitation Device having Dielectrophoresis Particle Separating Module
JP2022507573A (en) 2018-11-14 2022-01-18 プレジデント アンド フェロウズ オヴ ハーヴァード カレッジ Multiplexing of highly evolving virus variants with SHERLOCK
SG11202105083XA (en) 2018-11-14 2021-06-29 Broad Inst Inc Crispr system based droplet diagnostic systems and methods
US11459607B1 (en) 2018-12-10 2022-10-04 10X Genomics, Inc. Systems and methods for processing-nucleic acid molecules from a single cell using sequential co-partitioning and composite barcodes
US20250043366A1 (en) 2018-12-13 2025-02-06 The Broad Institute, Inc. Tiled assays using crispr-cas based detection
AU2019398307A1 (en) 2018-12-13 2021-06-10 Dna Script Direct oligonucleotide synthesis on cells and biomolecules
WO2020131586A2 (en) 2018-12-17 2020-06-25 The Broad Institute, Inc. Methods for identifying neoantigens
WO2020132010A1 (en) * 2018-12-20 2020-06-25 Alveo Technologies, Inc. Methods and compositions for detection of amplification products
US11473136B2 (en) 2019-01-03 2022-10-18 Ncan Genomics, Inc. Linked target capture
EP3908676A4 (en) * 2019-01-07 2023-02-01 Elegen Corporation Methods of using microfluidic positional encoding devices
US12169198B2 (en) 2019-01-08 2024-12-17 10X Genomics, Inc. Systems and methods for sample analysis
US11845983B1 (en) 2019-01-09 2023-12-19 10X Genomics, Inc. Methods and systems for multiplexing of droplet based assays
US11389799B2 (en) 2019-01-17 2022-07-19 The Regents Of The University Of Michigan Microfluidic device for size and deformability measurements and applications thereof
WO2020159543A1 (en) * 2019-02-01 2020-08-06 Hewlett-Packard Development Company, L.P. Cell marking systems
US11467153B2 (en) 2019-02-12 2022-10-11 10X Genomics, Inc. Methods for processing nucleic acid molecules
WO2020167866A1 (en) 2019-02-12 2020-08-20 10X Genomics, Inc. Systems and methods for transposon loading
EP4674961A2 (en) 2019-02-12 2026-01-07 10X Genomics, Inc. Methods for processing nucleic acid molecules
US12305239B2 (en) 2019-02-12 2025-05-20 10X Genomics, Inc. Analysis of nucleic acid sequences
US12275993B2 (en) 2019-02-12 2025-04-15 10X Genomics, Inc. Analysis of nucleic acid sequences
US11851683B1 (en) 2019-02-12 2023-12-26 10X Genomics, Inc. Methods and systems for selective analysis of cellular samples
WO2020167862A1 (en) 2019-02-12 2020-08-20 10X Genomics, Inc. Systems and methods for transfer of reagents between droplets
WO2020168258A1 (en) 2019-02-15 2020-08-20 Berkeley Lights, Inc. Laser-assisted repositioning of a micro-object and culturing of an attachment-dependent cell in a microfluidic environment
EP3698871A1 (en) 2019-02-19 2020-08-26 Gottfried Wilhelm Leibniz Universität Hannover Laser based sorting of droplets in microfluidic streams
US11655499B1 (en) 2019-02-25 2023-05-23 10X Genomics, Inc. Detection of sequence elements in nucleic acid molecules
US12000770B2 (en) * 2019-02-27 2024-06-04 Kyocera Corporation Particle separating and measuring device and particle separating and measuring apparatus
EP3938537A1 (en) 2019-03-11 2022-01-19 10X Genomics, Inc. Systems and methods for processing optically tagged beads
US10633693B1 (en) 2019-04-16 2020-04-28 Celsee Diagnostics, Inc. System and method for leakage control in a particle capture system
WO2020215011A1 (en) 2019-04-18 2020-10-22 Abs Global, Inc. System and process for continuous addition of cryoprotectant
WO2020218553A1 (en) * 2019-04-26 2020-10-29 ｂｉｔＢｉｏｍｅ株式会社 Digital analysis of microbial flora
JPWO2020218557A1 (en) * 2019-04-26 2020-10-29
WO2020223578A1 (en) * 2019-04-30 2020-11-05 Northeastern University Microfluidic chip for single cell pairing
WO2020222802A1 (en) * 2019-04-30 2020-11-05 Hewlett-Packard Development Company, L.P. Microfluidic concentrating particlizers
CN116174068A (en) 2019-04-30 2023-05-30 伯克利之光生命科技公司 Methods for Encapsulating and Assaying Cells
US12399150B2 (en) 2019-05-02 2025-08-26 The Board Of Trustees Of The University Of Illinois Atomic-to-nanoscale matter emission / flow regulation device
EP3966307B1 (en) 2019-05-07 2024-11-20 Bio-Rad Laboratories, Inc. System and method for automated single cell processing
US11273439B2 (en) 2019-05-07 2022-03-15 Bio-Rad Laboratories, Inc. System and method for target material retrieval from microwells
AU2020280104A1 (en) 2019-05-22 2022-01-20 Mission Bio, Inc. Method and apparatus for simultaneous targeted sequencing of DNA, RNA and protein
CN118291246A (en) 2019-06-14 2024-07-05 伯乐实验室有限公司 Systems and methods for automated single cell processing and analysis
EP3756763A1 (en) 2019-06-28 2020-12-30 IMEC vzw Microfluidic device
US11667954B2 (en) 2019-07-01 2023-06-06 Mission Bio, Inc. Method and apparatus to normalize quantitative readouts in single-cell experiments
DK182109B1 (en) * 2019-08-02 2025-08-18 Samplix Aps System and method for amplifying nucleic acids from single cells
EP4010111A4 (en) * 2019-08-05 2023-08-16 Academia Sinica MICROREACTOR AND METHOD OF USE
WO2021044245A1 (en) * 2019-09-04 2021-03-11 King Abdullah University Of Science And Technology System and method for determining demulsifier for separating water from water emulsion
US12235262B1 (en) 2019-09-09 2025-02-25 10X Genomics, Inc. Methods and systems for single cell protein analysis
US12297426B2 (en) 2019-10-01 2025-05-13 The Broad Institute, Inc. DNA damage response signature guided rational design of CRISPR-based systems and therapies
GB201914537D0 (en) 2019-10-08 2019-11-20 Univ Southampton Transcript analysis
CA3157359A1 (en) 2019-10-10 2021-04-15 1859, Inc. Methods and systems for microfluidic screening
CN112710585B (en) * 2019-10-25 2023-02-07 南京工程学院 A Method for Evaluating the Dynamic Stability of Emulsion Explosive Mixed on Site
US11857981B2 (en) 2019-12-23 2024-01-02 10X Genomics, Inc. Magnetic separator for an automated single cell sequencing system
JP2023504836A (en) * 2019-12-06 2023-02-07 バイオ－ラッド ラボラトリーズ インコーポレイテッド Sample processing barcoded bead compositions, methods, manufacturing methods and systems
WO2021119201A1 (en) 2019-12-10 2021-06-17 Enumerix, Inc. Methods and systems for three-dimensional lightsheet imaging
WO2021127576A1 (en) * 2019-12-20 2021-06-24 Berkeley Lights, Inc. Methods of penning micro-objects using positive dielectrophoresis
WO2021142133A1 (en) * 2020-01-07 2021-07-15 Elegen Corporation Dna assembly in microfluidics device having integrated solid-phase columns
US11628439B2 (en) 2020-01-13 2023-04-18 Abs Global, Inc. Single-sheath microfluidic chip
GB202001051D0 (en) * 2020-01-24 2020-03-11 Lightcast Discovery Ltd Methods and apparatus for high throughput microdroplet manipulation
TW202141019A (en) 2020-01-24 2021-11-01 美商史林夏特生化公司 Compositions and methods for cell-like calibration particles
WO2021154927A1 (en) * 2020-01-29 2021-08-05 Massachusetts Institute Of Technology Systems and methods for the injection of viscous fluids
USD993443S1 (en) * 2020-02-04 2023-07-25 Ut-Battelle, Llc Microfluidic glass chip interface bracket
USD989342S1 (en) * 2020-02-04 2023-06-13 Ut-Battelle, Llc Microfluidic polymer chip interface bracket
US12449419B1 (en) 2020-02-12 2025-10-21 10X Genomics, Inc. Methods for detecting binding of peptide-MHC monomers to T cells
CN115428088A (en) 2020-02-13 2022-12-02 10X基因组学有限公司 Systems and methods for joint interactive visualization of gene expression and DNA chromatin accessibility
GB202002077D0 (en) * 2020-02-14 2020-04-01 Nuclera Nucleics Ltd Methods for cell-free protein expression
KR102479966B1 (en) * 2020-02-20 2022-12-23 서울대학교산학협력단 Method for manufacturing organic solvent-free liposome using micro-fluidic chip
US11504719B2 (en) 2020-03-12 2022-11-22 Bio-Rad Laboratories, Inc. System and method for receiving and delivering a fluid for sample processing
KR102408166B1 (en) * 2020-04-02 2022-06-10 아주대학교산학협력단 Biosensing chip to analyze cell-derived materials during cell culture, and method for quantitative analysis of cell activity using the same
US12437398B2 (en) 2020-04-29 2025-10-07 Georgia Tech Research Corporation Systems and methods for specimen processing and storage
AU2021266702A1 (en) 2020-05-04 2022-11-10 Slingshot Biosciences, Inc. Compositions and methods for passive optical barcoding for multiplexed assays
GB202006525D0 (en) 2020-05-04 2020-06-17 Sphere Fluidics Ltd Surfactant
US11851700B1 (en) 2020-05-13 2023-12-26 10X Genomics, Inc. Methods, kits, and compositions for processing extracellular molecules
CN111624180B (en) * 2020-05-22 2021-06-22 西安交通大学 Cryofluorescence microscopy imaging system and method for capturing the morphology of moving macromolecules in microchannels
EP4171818A4 (en) 2020-06-30 2024-07-10 Plexium, Inc. FLUIDIC DEVICE AND METHOD
US12508586B2 (en) 2020-06-30 2025-12-30 Plexium, Inc. Uniform fluid flow with constant resistance and velocity
US11684920B2 (en) 2020-07-07 2023-06-27 International Business Machines Corporation Electrical tracking of a multiphase microfluidic flow
WO2022046079A1 (en) * 2020-08-28 2022-03-03 Hewlett-Packard Development Company, L.P. Barriers in microfluidic channels
WO2022072363A1 (en) * 2020-09-29 2022-04-07 The Regents Of The University Of California Method for rapid and large-scale generation of droplets and droplet libraries
US11655144B2 (en) * 2020-09-29 2023-05-23 Jiangsu University Method for preparing micro-cavity array surface product with inclined smooth bottom surface based on air molding method
EP4232601A4 (en) 2020-10-21 2024-10-09 ABS Global, Inc. Methods and systems for processing genetic samples to determine identity or detect contamination
US12084715B1 (en) 2020-11-05 2024-09-10 10X Genomics, Inc. Methods and systems for reducing artifactual antisense products
US12480158B1 (en) 2020-11-05 2025-11-25 10X Genomics, Inc. Methods and systems for processing polynucleotides
EP4248190A4 (en) 2020-11-23 2024-12-25 ABS Global, Inc. Modular flow cytometry systems and methods of processing samples
CN112578111B (en) * 2020-12-25 2024-06-18 扬州初心科技服务有限公司 Food antibiotic peristaltic multi-linked immunity detector
US12398262B1 (en) 2021-01-22 2025-08-26 10X Genomics, Inc. Triblock copolymer-based cell stabilization and fixation system and methods of use thereof
US12404177B2 (en) 2021-02-18 2025-09-02 University Of Connecticut Exfoliation of graphite to graphene by interface stabilization
CN117015617B (en) 2021-02-23 2025-04-04 10X基因组学有限公司 Probe-based nucleic acid and protein analysis
CN117241878A (en) 2021-03-05 2023-12-15 伊努梅里斯公司 Systems and methods for generating droplets and performing digital analysis
US12461029B2 (en) * 2021-03-10 2025-11-04 Northeastern University Continuous flow air sampling and rapid pathogen detection system
EP4323116A1 (en) * 2021-04-15 2024-02-21 ScreenSYS GmbH Systems and methods for microscopic object handling
JP2024516637A (en) 2021-04-26 2024-04-16 ザ ブリガム アンド ウィメンズ ホスピタル インコーポレイテッド Compositions and methods for characterizing alterations in polynucleotide sequences
WO2022231587A1 (en) * 2021-04-28 2022-11-03 Hewlett-Packard Development Company, L.P. Vibration of microfluidic device
GB202109969D0 (en) * 2021-07-09 2021-08-25 Lightcast Discovery Ltd Improvements in or relating to a method or an apparatus for detecting an interaction between a biological entity and a molecule
US12252745B2 (en) 2021-09-02 2025-03-18 Enumerix, Inc. Detection and digital quantitation of multiple targets
WO2023039389A1 (en) * 2021-09-07 2023-03-16 University Of Florida Research Foundation, Incorporated Efficient high-throughput electroporation for ev and exosome cargo loading
EP4423250A1 (en) 2021-10-29 2024-09-04 Slingshot Biosciences, Inc. Hydrogel particles as feeder cells and as synthetic antigen presenting cells
CN113952903A (en) * 2021-11-17 2022-01-21 上海东富龙拓溥科技有限公司 A kind of preparation method of medicinal microcapsules
IT202100030557A1 (en) * 2021-12-02 2023-06-02 Istituto Naz Di Astrofisica METHOD AND RELATED SYSTEM FOR THE DETECTION OF A VIRAL AGENT BY MEANS OF MICROWAVE DIELECTRIC SPECTROSCOPY
US12465910B2 (en) 2021-12-10 2025-11-11 Bio-Rad Laboratories, Inc. Compositions, methods, and systems for sample processing with morphology-adjustable functionalized particles
US12253467B2 (en) * 2021-12-13 2025-03-18 Saudi Arabian Oil Company Determining partition coefficients of tracer analytes
CN118922556A (en) 2021-12-20 2024-11-08 伊努梅里斯公司 Detection and digital quantification of multiple targets
WO2023147334A1 (en) * 2022-01-25 2023-08-03 Berkeley Lights, Inc. Methods of assaying micro-objects in a microfluidic device
US20230241604A1 (en) * 2022-01-31 2023-08-03 Tdk Corporation Methods and Devices for Measuring Particle Properties
KR20250018496A (en) 2022-05-05 2025-02-06 슬링샷 바이오사이언시즈 인코포레이티드 Engineered particles as erythrocyte mimics for hematology and compositions containing same
CA3257621A1 (en) 2022-06-02 2023-12-07 Slingshot Biosciences, Inc. Apoptotic cell mimic
US20260016432A1 (en) * 2022-07-25 2026-01-15 Interface Fluidics Ltd. Microfluidic devices, microfluidic systems, and methods for assessing thermophysical properties of a fluid
WO2024064914A1 (en) * 2022-09-22 2024-03-28 Sigil Biosciences, Inc. Devices and systems for isolating particles in solution by particle permitivity
WO2024092161A2 (en) 2022-10-26 2024-05-02 Slingshot Biosciences, Inc. Size-tunable synthetic particles with tunable optical properties and methods for using the same for immune cell activation
WO2024096137A1 (en) * 2022-11-04 2024-05-10 国立大学法人東京大学 Droplet separation device, separation signal generation device, separation signal generation method, and program
WO2024097393A1 (en) * 2022-11-04 2024-05-10 Fluid Discovery Systems, compositions, and methods for single cell analysis
WO2024137514A1 (en) 2022-12-22 2024-06-27 Synthego Corporation Systems and method for automated oligonucleotide synthesis
WO2024159194A1 (en) * 2023-01-27 2024-08-02 University Of Tennessee Research Foundation Diamond-based microfluidic alpha spectrometer
EP4662478A1 (en) * 2023-02-10 2025-12-17 IDEXX Laboratories, Inc. Methods, systems, and devices for linear electrowetting cartridges
US12531162B1 (en) * 2023-05-31 2026-01-20 Northeastern University Multi-dimensional phenotypic space for genotype to phenotype mapping and intelligent design of cancer drug therapies using a deep learning net
US12461007B2 (en) 2023-08-29 2025-11-04 Slingshot Biosciences, Inc. CD34 stem cell mimics
WO2025064904A1 (en) * 2023-09-22 2025-03-27 Astrabios Inc. Integrated digital pcr systems
WO2025097023A1 (en) * 2023-11-03 2025-05-08 University Of Utah Research Foundation Electrokinetic assessment of critical quality attributes of nanoparticles
GB2636109A (en) 2023-11-29 2025-06-11 Sphere Fluidics Ltd Dendronized surfactants
CN118179622B (en) * 2024-04-01 2025-09-05 上海澎赞生物科技有限公司 A multi-core emulsion droplet preparation chip, modification method and emulsion droplet preparation system
CN118744020B (en) * 2024-07-31 2025-09-23 武汉理工大学 Microfluidic chip and device for preparing thermally induced rapid-setting microcapsules
TWI904887B (en) * 2024-10-09 2025-11-11 國立虎尾科技大學 Microfluidic detecting system and method for detecting cell
CN120748587B (en) * 2025-09-05 2025-10-31 山西医科大学 A method and system for controlling the functionality of nanopores

Citations (1033)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2097692A (en) 1936-03-23 1937-11-02 Bohn Aluminium & Brass Corp Method and machine for forming bearing shells
US2164172A (en) 1938-04-30 1939-06-27 Gen Electric Liquid-dispensing apparatus
US2636855A (en) 1948-03-25 1953-04-28 Hilger & Watts Ltd Method of producing photoconductive coatings
US2656508A (en) 1949-08-27 1953-10-20 Wallace H Coulter Means for counting particles suspended in a fluid
US2692800A (en) 1951-10-08 1954-10-26 Gen Electric Nozzle flow control
US2797149A (en) 1953-01-08 1957-06-25 Technicon International Ltd Methods of and apparatus for analyzing liquids containing crystalloid and non-crystalloid constituents
US2879141A (en) 1955-11-16 1959-03-24 Technicon Instr Automatic analyzing apparatus
US2971700A (en) 1957-07-22 1961-02-14 Vilbiss Co Apparatus for coating articles with chemically reactive liquids
GB1148543A (en) 1966-01-10 1969-04-16 Bachofen Willy A Sight glass fitting for optical flow supervision
US3479141A (en) 1967-05-17 1969-11-18 Technicon Corp Method and apparatus for analysis
US3608821A (en) 1965-10-15 1971-09-28 Agfa Gevaert Ag Electrostatic atomization of liquids
US3621059A (en) 1969-07-30 1971-11-16 Du Pont Amides of hexafluoropropylene oxide polymer acids and polyalklene oxide
DE2100685A1 (en) 1971-01-08 1972-07-20 Badische Anilin- & Soda-Fabrik Ag, 6700 Ludwigshafen Pure 4-amino-5-halo-6-pyridazones sepn - from isomers by extraction with halohydrocarbons, alkanols or ethers
US3698635A (en) 1971-02-22 1972-10-17 Ransburg Electro Coating Corp Spray charging device
US3784471A (en) 1970-05-11 1974-01-08 Avco Corp Solid additives dispersed in perfluorinated liquids with perfluoroalkyl ether dispersants
US3816331A (en) 1972-07-05 1974-06-11 Ncr Continuous encapsulation and device therefor
US3828085A (en) 1970-07-09 1974-08-06 Allied Chem Novel amidoamine oxides
CH563087A5 (en) 1972-10-06 1975-06-13 Westinghouse Electric Corp
CH563807A5 (en) 1973-02-14 1975-07-15 Battelle Memorial Institute Fine granules and microcapsules mfrd. from liquid droplets - partic. of high viscosity requiring forced sepn. of droplets
US3930061A (en) 1974-04-08 1975-12-30 Ransburg Corp Electrostatic method for forming structures and articles
US3960187A (en) 1974-07-23 1976-06-01 Usm Corporation Method and device for metering and dispersing fluid materials
GB1446998A (en) 1974-02-25 1976-08-18 Sauter Ag Apparatus for mixing at least two fluent media
US3980541A (en) 1967-06-05 1976-09-14 Aine Harry E Electrode structures for electric treatment of fluids and filters using same
US3982541A (en) 1974-07-29 1976-09-28 Esperance Jr Francis A L Eye surgical instrument
US4014469A (en) 1975-11-17 1977-03-29 Kozo Sato Nozzle of gas cutting torch
US4022575A (en) 1974-09-16 1977-05-10 Block Engineering, Inc. Automatic chemical analyzer
US4034966A (en) 1975-11-05 1977-07-12 Massachusetts Institute Of Technology Method and apparatus for mixing particles
US4059552A (en) 1974-06-21 1977-11-22 The Dow Chemical Company Cross-linked water-swellable polymer particles
US4091042A (en) 1977-08-19 1978-05-23 American Cyanamid Company Continuous adiabatic process for the mononitration of benzene
JPS5372016A (en) 1976-12-08 1978-06-27 Toyo Tire & Rubber Co Ltd Apparatus for preparation and supply of heavy oil w/o emulsion fuel
US4117550A (en) 1977-02-14 1978-09-26 Folland Enertec Ltd. Emulsifying system
US4130394A (en) 1977-10-03 1978-12-19 Technicon Instruments Corporation Short sample detection
US4210809A (en) 1979-03-16 1980-07-01 Technicon Instruments Corporation Method and apparatus for the non-invasive determination of the characteristics of a segmented fluid stream
US4253846A (en) 1979-11-21 1981-03-03 Technicon Instruments Corporation Method and apparatus for automated analysis of fluid samples
JPS5636053A (en) 1979-08-28 1981-04-09 Bifok Ab Method of continuous flowing analysis
US4266721A (en) 1979-09-17 1981-05-12 Ppg Industries, Inc. Spray application of coating compositions utilizing induction and corona charging means
US4279345A (en) 1979-08-03 1981-07-21 Allred John C High speed particle sorter using a field emission electrode
US4297345A (en) 1975-04-14 1981-10-27 Beecham Group Limited Antibacterial agents
GB2097692A (en) 1981-01-10 1982-11-10 Shaw Stewart P D Combining chemical reagents
BR8200642A (en) 1981-02-06 1982-12-14 Honda Motor Co Ltd APPLIANCE FOR WELDING
US4378957A (en) 1978-08-11 1983-04-05 Malkin Daniel D Reduction gear of electronic wristwatch with stepping motor and sweep second hand
US4383767A (en) 1979-11-05 1983-05-17 Agency Of Industrial Science & Technology Method for blending by combining fine particles
JPS5949832A (en) 1982-08-14 1984-03-22 バイエル・アクチエンゲゼルシヤフト Dispersion manufacturing method and device
US4439980A (en) 1981-11-16 1984-04-03 The United States Of America As Represented By The Secretary Of The Navy Electrohydrodynamic (EHD) control of fuel injection in gas turbines
JPS59102163A (en) 1982-11-15 1984-06-13 テクニコン，インストルメンツ，コ−ポレ−シヨン Continuous flowing method, continuous flowing device and single flow-path continuous flow analyzer
EP0047130B1 (en) 1980-08-28 1985-02-13 E.I. Du Pont De Nemours And Company Flow analysis
US4508265A (en) 1981-06-18 1985-04-02 Agency Of Industrial Science & Technology Method for spray combination of liquids and apparatus therefor
US4533634A (en) 1983-01-26 1985-08-06 Amf Inc. Tissue culture medium
US4566908A (en) 1984-02-24 1986-01-28 Mita Industrial Company, Limited Azoic pigments having a silica core
US4585209A (en) 1983-10-27 1986-04-29 Harry E. Aine Miniature valve and method of making same
US4618476A (en) 1984-02-10 1986-10-21 Eastman Kodak Company Capillary transport device having speed and meniscus control means
US4675285A (en) 1984-09-19 1987-06-23 Genetics Institute, Inc. Method for identification and isolation of DNA encoding a desired protein
US4676274A (en) 1985-02-28 1987-06-30 Brown James F Capillary flow control
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
EP0249007A2 (en) 1986-04-14 1987-12-16 The General Hospital Corporation A method of screening hybridomas
US4739044A (en) 1985-06-13 1988-04-19 Amgen Method for derivitization of polynucleotides
US4757141A (en) 1985-08-26 1988-07-12 Applied Biosystems, Incorporated Amino-derivatized phosphite and phosphate linking agents, phosphoramidite precursors, and useful conjugates thereof
US4767515A (en) 1987-07-30 1988-08-30 The United States Of America As Represented By The United States Department Of Energy Surface area generation and droplet size control in solvent extraction systems utilizing high intensity electric fields
US4767929A (en) 1986-10-06 1988-08-30 The United States Of America As Represented By The United State Department Of Energy Extended range radiation dose-rate monitor
US4779805A (en) 1982-10-13 1988-10-25 Imperial Chemical Industries Plc Electrostatic sprayhead assembly
US4795330A (en) 1986-02-21 1989-01-03 Imperial Chemical Industries Plc Apparatus for particles
US4801529A (en) 1985-06-18 1989-01-31 Brandeis University Methods for isolating mutant microoganisms using microcapsules coated with indicator material
US4801086A (en) 1985-02-19 1989-01-31 Imperial Chemical Industries Plc Spraying apparatus
GB2210532A (en) 1987-09-30 1989-06-07 Sony Corp Digital signal processing circuit for video camera
US4853336A (en) 1982-11-15 1989-08-01 Technicon Instruments Corporation Single channel continuous flow system
US4856363A (en) 1988-02-10 1989-08-15 Wickes Manufacturing Company Parking brake assembly
US4859363A (en) 1985-02-26 1989-08-22 I.S.C. Chemicals Limited Emulsions of perfluorocarbons in aqueous media
US4865444A (en) 1984-04-05 1989-09-12 Mobil Oil Corporation Apparatus and method for determining luminosity of hydrocarbon fuels
US4883750A (en) 1984-12-13 1989-11-28 Applied Biosystems, Inc. Detection of specific sequences in nucleic acids
US4908112A (en) 1988-06-16 1990-03-13 E. I. Du Pont De Nemours & Co. Silicon semiconductor wafer for analyzing micronic biological samples
US4931225A (en) 1987-12-30 1990-06-05 Union Carbide Industrial Gases Technology Corporation Method and apparatus for dispersing a gas into a liquid
US4941959A (en) 1989-11-27 1990-07-17 Martin Marietta Energy Systems, Inc. Electric field-driven, magnetically-stabilized ferro-emulsion phase contactor
US4962885A (en) 1978-04-17 1990-10-16 Coffee Ronald A Process and apparatus for spraying liquid
US4963498A (en) 1985-08-05 1990-10-16 Biotrack Capillary flow device
EP0402995A2 (en) 1989-06-12 1990-12-19 Johnson &amp; Johnson Clinical Diagnostics, Inc. Temperature control device and reaction vessel
WO1990015807A1 (en) 1989-06-22 1990-12-27 Applications Et Transferts De Technologies Avancées Fluorine and phosphorous-containing amphiphilic molecules with surfactant properties
US4981580A (en) 1989-05-01 1991-01-01 Coulter Corporation Coincidence arbitration in a flow cytomery sorting system
FR2650657A1 (en) 1989-08-05 1991-02-08 Scras Sa APPARATUS FOR THE AUTOMATIC AND REPEATED EXECUTION OF A THERMAL CYCLE FOR THE TREATMENT OF BIOLOGICAL SAMPLES
EP0418635A1 (en) 1989-09-12 1991-03-27 Biotest AG Enzyme immunoassay using peroxidase as the marker enzyme
WO1991005058A1 (en) 1989-10-05 1991-04-18 Glenn Kawasaki Cell-free synthesis and isolation of novel genes and polypeptides
WO1991007772A1 (en) 1989-11-17 1991-05-30 Charged Injection Corporation Methods and apparatus for dispersing a fluent material utilizing an electron beam
US5055390A (en) 1988-04-22 1991-10-08 Massachusetts Institute Of Technology Process for chemical manipulation of non-aqueous surrounded microdroplets
JPH03232525A (en) 1990-02-07 1991-10-16 Gunma Univ Formation of uniform liquid drop
WO1991016966A1 (en) 1990-05-10 1991-11-14 Pharmacia Biosensor Ab Microfluidic structure and process for its manufacture
US5091652A (en) 1990-01-12 1992-02-25 The Regents Of The University Of California Laser excited confocal microscope fluorescence scanner and method
WO1992003734A1 (en) 1990-08-20 1992-03-05 Alain De Weck A method for measuring t-lymphocyte responses by chemiluminescent assays
US5096615A (en) 1988-07-19 1992-03-17 The United States Of America As Represented By The United States Department Of Energy Solid aerosol generator
EP0476178A1 (en) 1990-09-21 1992-03-25 Bioplex Medical B.V. Device for placing styptic material on perforated blood vessels
US5104813A (en) 1989-04-13 1992-04-14 Biotrack, Inc. Dilution and mixing cartridge
FR2669028A1 (en) 1990-11-13 1992-05-15 Rhone Poulenc Chimie PROCESS FOR THE PRODUCTION OF RARE EARTH DUAL OXALATES AND AMMONIUM AND THEIR USES FOR THE MANUFACTURE OF RARE EARTH OXIDES.
US5122360A (en) 1989-11-27 1992-06-16 Martin Marietta Energy Systems, Inc. Method and apparatus for the production of metal oxide powder
US5149625A (en) 1987-08-11 1992-09-22 President And Fellows Of Harvard College Multiplex analysis of DNA
WO1992021746A1 (en) 1991-05-30 1992-12-10 Center For Blood Research, Inc. Device and method for the analysis of rolling blood leukocytes and identifying inhibitors and promoters
US5180662A (en) 1988-01-05 1993-01-19 The United States Of America As Represented By The Department Of Health And Human Services Cytotoxic T lymphocyte activation assay
US5185099A (en) 1988-04-20 1993-02-09 Institut National De Recherche Chimique Appliquee Visco-elastic, isotropic materials based on water, fluorinate sufactants and fluorinated oils, process for their preparation, and their use in various fields, such as optics, pharmacology and electrodynamics
IE922432A1 (en) 1991-08-08 1993-02-10 Tioxide Specialties Ltd Preparation of Titanium Derivatives
WO1993003151A1 (en) 1991-08-10 1993-02-18 Medical Research Council Treatment of cell populations
US5188291A (en) 1989-05-31 1993-02-23 Her Majesty The Queen In Right Of New Zealand Fluid distribution device
US5188290A (en) 1990-02-16 1993-02-23 J. Wagner Gmbh Electrostatic compressed air paint spray gun
US5192659A (en) 1989-08-25 1993-03-09 Genetype Ag Intron sequence analysis method for detection of adjacent and remote locus alleles as haplotypes
US5204112A (en) 1986-06-16 1993-04-20 The Liposome Company, Inc. Induction of asymmetry in vesicles
WO1993008278A1 (en) 1991-10-16 1993-04-29 Affymax Technologies N.V. Peptide library and screening method
US5207973A (en) 1989-11-27 1993-05-04 Martin Marietta Energy Systems, Inc. Method and apparatus for the production of metal oxide powder
EP0546174A1 (en) 1991-06-29 1993-06-16 Miyazaki-Ken Monodisperse single and double emulsions and production thereof
US5241159A (en) 1992-03-11 1993-08-31 Eastman Kodak Company Multi-zone heating for a fuser roller
WO1993022421A1 (en) 1992-05-01 1993-11-11 Trustees Of The University Of Pennsylvania Microfabricated sperm handling devices
US5262027A (en) 1991-03-22 1993-11-16 Martin Marietta Energy Systems, Inc. Method of using an electric field controlled emulsion phase contactor
US5270163A (en) 1990-06-11 1993-12-14 University Research Corporation Methods for identifying nucleic acid ligands
JPH0665609A (en) 1992-08-25 1994-03-08 Mitsubishi Materials Corp Manufacturing method of iron-based sintered forged body
US5296375A (en) 1992-05-01 1994-03-22 Trustees Of The University Of Pennsylvania Mesoscale sperm handling devices
US5304487A (en) 1992-05-01 1994-04-19 Trustees Of The University Of Pennsylvania Fluid handling in mesoscale analytical devices
US5310653A (en) 1989-10-24 1994-05-10 Board Of Regents, The University Of Texas System Tumor marker protein and antibodies thereto for cancer risk assessment or diagnosis
US5313009A (en) 1990-01-04 1994-05-17 Nrm International Technologies C.V. Nitration process
WO1994016332A1 (en) 1993-01-13 1994-07-21 Yeda Research And Development Co. Ltd. Method for screening catalytic non-enzyme polypeptides and proteins
US5333675A (en) 1986-02-25 1994-08-02 Hoffmann-La Roche Inc. Apparatus and method for performing automated amplification of nucleic acid sequences and assays using heating and cooling steps
US5344594A (en) 1991-10-29 1994-09-06 Xerox Corporation Method for the fabrication of multicolored balls for a twisting ball display
US5344489A (en) 1991-11-15 1994-09-06 Manfred R. Kuehnle Synthetic, monodispersed color pigments for the coloration of media such as printing inks, and method and apparatus for making same
JPH06265447A (en) 1993-03-16 1994-09-22 Hitachi Ltd Microreactor and trace component measuring device using the same
EP0618001A1 (en) 1993-03-31 1994-10-05 Rhone-Poulenc Nutrition Animale Process for preparing active agents containing spherules
US5354670A (en) 1991-12-24 1994-10-11 The President And Fellows Of Harvard College Site-directed mutagenesis of DNA
EP0620432A1 (en) 1993-04-15 1994-10-19 Ciba-Geigy Ag Method for controlling sample introduction in microcolumn separation techniques and sampling device
WO1994024314A1 (en) 1993-04-19 1994-10-27 Kauffman Stuart A Random chemistry for the generation of new compounds
WO1994023738A1 (en) 1993-04-19 1994-10-27 Medisorb Technologies International L.P. Encapsulation of nucleic acids with conjugates that facilitate and target cellular uptake and gene expression
WO1994026766A1 (en) 1993-02-19 1994-11-24 Barnes Wayne M Dna polymerases with enhanced thermostability and enhanced length and efficiency of primer extension
US5397605A (en) 1992-05-29 1995-03-14 Barbieri; Girolamo Method and apparatus for electrostatically coating a workpiece with paint
US5399461A (en) 1987-08-21 1995-03-21 Sharp Kabushiki Kaisha Optical disk for use in optical memory devices
US5399491A (en) 1989-07-11 1995-03-21 Gen-Probe Incorporated Nucleic acid sequence amplification methods
US5403617A (en) 1993-09-15 1995-04-04 Mobium Enterprises Corporation Hybrid pulsed valve for thin film coating and method
WO1995011922A1 (en) 1993-10-29 1995-05-04 Affymax Technologies N.V. In vitro peptide and antibody display libraries
US5413924A (en) 1992-02-13 1995-05-09 Kosak; Kenneth M. Preparation of wax beads containing a reagent for release by heating
US5417235A (en) 1993-07-28 1995-05-23 Regents Of The University Of Michigan Integrated microvalve structures with monolithic microflow controller
WO1995019922A1 (en) 1994-01-25 1995-07-27 Ingenieurbüro Von Borries Strip packing
US5445934A (en) 1989-06-07 1995-08-29 Affymax Technologies N.V. Array of oligonucleotides on a solid substrate
WO1995024929A2 (en) 1994-03-15 1995-09-21 Brown University Research Foundation Polymeric gene delivery system
US5452878A (en) 1991-06-18 1995-09-26 Danfoss A/S Miniature actuating device
US5452955A (en) 1992-06-25 1995-09-26 Vattenfall Utvecking Ab Device for mixing two fluids having different temperatures
US5454472A (en) 1991-08-19 1995-10-03 Fraunhofer Gesellschaft Zur Forderung Der Angewandten Forschung E.V. Method of continuously separating mixtures of microscopic dielectric particles and apparatus for carrying through this method
US5460945A (en) 1991-05-30 1995-10-24 Center For Blood Research, Inc. Device and method for analysis of blood components and identifying inhibitors and promoters of the inflammatory response
US5468613A (en) 1986-03-13 1995-11-21 Hoffmann-La Roche Inc. Process for detecting specific nucleotide variations and genetic polymorphisms present in nucleic acids
US5475610A (en) 1990-11-29 1995-12-12 The Perkin-Elmer Corporation Thermal cycler for automatic performance of the polymerase chain reaction with close temperature control
US5475096A (en) 1990-06-11 1995-12-12 University Research Corporation Nucleic acid ligands
WO1995033447A1 (en) 1994-06-09 1995-12-14 Alliance Pharmaceutical Corp. Stable reverse and multiple fluorocarbon emulsions
US5486335A (en) 1992-05-01 1996-01-23 Trustees Of The University Of Pennsylvania Analysis based on flow restriction
US5498392A (en) 1992-05-01 1996-03-12 Trustees Of The University Of Pennsylvania Mesoscale polynucleotide amplification device and method
US5498523A (en) 1988-07-12 1996-03-12 President And Fellows Of Harvard College DNA sequencing with pyrophosphatase
US5503851A (en) 1992-07-10 1996-04-02 Ferring Arzneimittel Gmbh Microencapsulation of water-soluble medicaments
US5512131A (en) 1993-10-04 1996-04-30 President And Fellows Of Harvard College Formation of microstamped patterns on surfaces and derivative articles
US5516635A (en) 1991-10-15 1996-05-14 Ekins; Roger P. Binding assay employing labelled reagent
US5518709A (en) 1991-04-10 1996-05-21 Andaris Limited Preparation of diagnostic agents
NZ264353A (en) 1991-05-30 1996-05-28 For Blood Research Inc Centre Method of collecting or purifying leukocytes from a fluid sample, apparatus, immune response inhibitor test
US5523162A (en) 1990-04-03 1996-06-04 Ppg Industries, Inc. Water repellent surface treatment for plastic and coated plastic substrates
JPH08153669A (en) 1994-11-30 1996-06-11 Hitachi Ltd Thin film forming method and forming apparatus
WO1996034112A1 (en) 1995-04-24 1996-10-31 Chromaxome Corp. Methods for generating and screening novel metabolic pathways
WO1996038730A1 (en) 1995-06-02 1996-12-05 Cdc Technologies, Inc. Apparatus and method for mixing fluids for analysis
WO1996040057A2 (en) 1995-06-07 1996-12-19 Alliance Pharmaceutical Corp. Reverse fluorocarbon emulsion compositions for drug delivery
WO1996040062A1 (en) 1995-06-07 1996-12-19 Georgetown University A method of transfection of cells using liposomally encapsulated nucleic acids
WO1996040723A1 (en) 1995-06-07 1996-12-19 The General Hospital Corporation Catalytic dna
US5587128A (en) 1992-05-01 1996-12-24 The Trustees Of The University Of Pennsylvania Mesoscale polynucleotide amplification devices
US5589136A (en) 1995-06-20 1996-12-31 Regents Of The University Of California Silicon-based sleeve devices for chemical reactions
WO1997000442A1 (en) 1995-06-16 1997-01-03 The University Of Washington Microfabricated differential extraction device and method
WO1997000125A1 (en) 1995-06-16 1997-01-03 Novartis Ag Flow cell for the passive mixing of flowable substances
WO1997004297A1 (en) 1995-07-21 1997-02-06 Northeastern University Microscale fluid handling system
WO1997004748A2 (en) 1995-08-01 1997-02-13 Advanced Therapies, Inc. Enhanced artificial viral envelopes for cellular delivery of therapeutic substances
US5604097A (en) 1994-10-13 1997-02-18 Spectragen, Inc. Methods for sorting polynucleotides using oligonucleotide tags
US5610016A (en) 1994-03-30 1997-03-11 Mochida Pharmaceutical Co., Ltd. Method for measuring adenyl group-containing substances using heteropoly-acid
US5612188A (en) 1991-11-25 1997-03-18 Cornell Research Foundation, Inc. Automated, multicompartmental cell culture system
US5616478A (en) 1992-10-14 1997-04-01 Chetverin; Alexander B. Method for amplification of nucleic acids in solid media
US5617997A (en) 1994-06-13 1997-04-08 Praxair Technology, Inc. Narrow spray angle liquid fuel atomizers for combustion
DE4308839C2 (en) 1993-03-19 1997-04-30 Jordanow & Co Gmbh Device for mixing flow media
US5636400A (en) 1995-08-07 1997-06-10 Young; Keenan L. Automatic infant bottle cleaner
US5641658A (en) 1994-08-03 1997-06-24 Mosaic Technologies, Inc. Method for performing amplification of nucleic acid with two primers bound to a single solid support
US5643729A (en) 1994-02-24 1997-07-01 Boehringer Ingelheim International Gmbh Methods for diagnosing cancer, precancerous state, or susceptibility to other forms of diseases by detecting an acceleration of exon skipping in IRF-1 mRNA
WO1997023140A1 (en) 1995-12-18 1997-07-03 Abbott Laboratories Stabilization of liquid nutritional products comprising soy polysaccharide
WO1997028556A1 (en) 1996-01-31 1997-08-07 Hewlett-Packard Company Apparatus for forming liquid droplets having a mechanically fixed inner microtube
US5656493A (en) 1985-03-28 1997-08-12 The Perkin-Elmer Corporation System for automated performance of the polymerase chain reaction
US5655517A (en) 1994-03-29 1997-08-12 Electrosols, Ltd. Dispensing device
US5656155A (en) 1994-04-26 1997-08-12 Ip Holding Company Thermophilic aerobic waste treatment system
US5661222A (en) 1995-04-13 1997-08-26 Dentsply Research & Development Corp. Polyvinylsiloxane impression material
US5670325A (en) 1996-08-14 1997-09-23 Exact Laboratories, Inc. Method for the detection of clonal populations of transformed cells in a genomically heterogeneous cellular sample
WO1997038318A1 (en) 1996-04-04 1997-10-16 Novartis Ag Device for counting small particles and a sorting apparatus comprising such a device
WO1997040141A2 (en) 1996-04-25 1997-10-30 Medical Research Council Isolation of enzymes
WO1997039814A1 (en) 1996-04-19 1997-10-30 Central Research Laboratories Limited Method and apparatus for diffusive transfer between immiscible liquids
WO1997045644A1 (en) 1996-05-31 1997-12-04 The University Of Washington Valveless liquid microswitch
US5695934A (en) 1994-10-13 1997-12-09 Lynx Therapeutics, Inc. Massively parallel sequencing of sorted polynucleotides
WO1997047763A1 (en) 1996-06-14 1997-12-18 Curagen Corporation Identification and comparison of protein-protein interactions and inhibitors thereof
WO1998000231A1 (en) 1996-06-28 1998-01-08 Caliper Technologies Corporation High-throughput screening assay systems in microscale fluidic devices
CA2258481A1 (en) 1996-06-28 1998-01-08 Caliper Technologies Corporation Electropipettor and compensation means for electrophoretic bias
WO1998000705A1 (en) 1996-06-28 1998-01-08 Caliper Technologies Corporation Electropipettor and compensation means for electrophoretic bias
AU3499097A (en) 1996-06-28 1998-01-21 Caliper Technologies Corporation High throughput screening assay systems in microscale fluidic devices
WO1998002237A1 (en) 1996-07-15 1998-01-22 Kemgas Limited Production of powders
US5726026A (en) 1992-05-01 1998-03-10 Trustees Of The University Of Pennsylvania Mesoscale sample preparation device and systems for determination and processing of analytes
WO1998010267A1 (en) 1996-09-04 1998-03-12 Technical University Of Denmark A micro flow system for particle separation and analysis
US5733526A (en) 1995-12-14 1998-03-31 Alliance Pharmaceutical Corp. Hydrocarbon oil/fluorochemical preparations and methods of use
WO1998013502A2 (en) 1996-09-27 1998-04-02 Icos Corporation Method to identify compounds for disrupting protein/protein interactions
US5739036A (en) 1996-04-15 1998-04-14 Dade International Inc. Method for analysis
US5744366A (en) 1992-05-01 1998-04-28 Trustees Of The University Of Pennsylvania Mesoscale devices and methods for analysis of motile cells
US5750988A (en) 1994-07-11 1998-05-12 Hewlett-Packard Company Orthogonal ion sampling for APCI mass spectrometry
WO1998022625A1 (en) 1996-11-20 1998-05-28 The Regents Of The University Of Michigan Microfabricated isothermal nucleic acid amplification devices and methods
WO1998023733A2 (en) 1996-11-27 1998-06-04 University Of Washington Thermostable polymerases having altered fidelity
US5762775A (en) 1994-09-21 1998-06-09 Lockheed Martin Energy Systems, Inc. Method for electrically producing dispersions of a nonconductive fluid in a conductive medium
US5779868A (en) 1996-06-28 1998-07-14 Caliper Technologies Corporation Electropipettor and compensation means for electrophoretic bias
US5783431A (en) 1996-04-24 1998-07-21 Chromaxome Corporation Methods for generating and screening novel metabolic pathways
WO1998031700A1 (en) 1997-01-21 1998-07-23 The General Hospital Corporation Selection of proteins using rna-protein fusions
WO1998033001A1 (en) 1997-01-29 1998-07-30 The Board Of Trustees Of The Leland Stanford Junior University Micromachined fluidic coupler
US5789206A (en) 1995-07-07 1998-08-04 Myriad Genetics, Inc. Method for ligating adaptors to nucleic acids which methods are useful for obtaining the ends of genes
WO1998034120A1 (en) 1997-01-31 1998-08-06 Universite De Montreal Protein fragment complementation assays to detect biomolecular interactions
JPH10217477A (en) 1997-02-07 1998-08-18 Fuji Xerox Co Ltd Ink jet recording device
WO1998037186A1 (en) 1997-02-18 1998-08-27 Actinova Limited In vitro peptide or protein expression library
WO1998041869A1 (en) 1997-03-18 1998-09-24 Chromaxome Corporation Methods for screening compounds using encapsulated cells
US5813988A (en) 1995-02-03 1998-09-29 Research Foundation Time-resolved diffusion tomographic imaging in highly scattering turbid media
US5840506A (en) 1996-06-05 1998-11-24 Thomas Jefferson University Methods for the diagnosis and prognosis of cancer
WO1998052691A1 (en) 1997-05-16 1998-11-26 Alberta Research Council Microfluidic system and methods of use
US5846719A (en) 1994-10-13 1998-12-08 Lynx Therapeutics, Inc. Oligonucleotide tags for sorting and identification
US5849491A (en) 1995-09-22 1998-12-15 Terragen Diversity Inc. Method for isolating xylanase gene sequences from soil DNA, compositions useful in such method and compositions obtained thereby
US5851769A (en) 1995-09-27 1998-12-22 The Regents Of The University Of California Quantitative DNA fiber mapping
WO1998058085A1 (en) 1997-06-16 1998-12-23 Diversa Corporation High throughput screening for novel enzymes
US5858655A (en) 1991-03-11 1999-01-12 The General Hospital Corporation Method for diagnosing neoplasia by detecting expression of PRAD1 cyclin
US5858187A (en) 1996-09-26 1999-01-12 Lockheed Martin Energy Systems, Inc. Apparatus and method for performing electrodynamic focusing on a microchip
US5858670A (en) 1990-07-02 1999-01-12 The Arizona Board Of Regents Bio-oligomer libraries and a method of use thereof
WO1999002671A1 (en) 1997-07-07 1999-01-21 Medical Research Council In vitro sorting method
EP0895120A1 (en) 1997-07-15 1999-02-03 Konica Corporation Method for preparing silver halide emulsion
US5876771A (en) 1996-06-20 1999-03-02 Tetra Laval Holdings & Finance, Sa Process and article for determining the residence time of a food particle
US5882680A (en) 1995-12-07 1999-03-16 Freund Industrial Co., Ltd. Seamless capsule and method of manufacturing the same
US5882856A (en) 1995-06-07 1999-03-16 Genzyme Corporation Universal primer sequence for multiplex DNA amplification
US5884846A (en) 1996-09-19 1999-03-23 Tan; Hsiaoming Sherman Pneumatic concentric nebulizer with adjustable and capillaries
US5887755A (en) 1995-06-06 1999-03-30 Quantic Biomedical Partners Wound sealant preparation and application device and method
US5888778A (en) 1997-06-16 1999-03-30 Exact Laboratories, Inc. High-throughput screening method for identification of genetic mutations or disease-causing microorganisms using segmented primers
US5888746A (en) 1994-06-10 1999-03-30 Institute Of Molecular And Cell Biology Tumor diagnosis and prognosis
WO1999022858A1 (en) 1997-11-05 1999-05-14 British Nuclear Fuels Plc Reactions of aromatic compounds
WO1999028507A1 (en) 1997-12-04 1999-06-10 Exact Laboratories, Inc. Methods for the detection of chromosomal aberrations
WO1999028020A1 (en) 1997-12-01 1999-06-10 Minnesota Mining And Manufacturing Company Process for production of heat sensitive dispersions or emulsions
WO1999031019A1 (en) 1997-12-17 1999-06-24 Universidad De Sevilla Device and method for creating spherical particles of uniform size
US5921678A (en) 1997-02-05 1999-07-13 California Institute Of Technology Microfluidic sub-millisecond mixers
US5928870A (en) 1997-06-16 1999-07-27 Exact Laboratories, Inc. Methods for the detection of loss of heterozygosity
US5935331A (en) 1994-09-09 1999-08-10 Matsushita Electric Industrial Co., Ltd. Apparatus and method for forming films
US5942443A (en) 1996-06-28 1999-08-24 Caliper Technologies Corporation High throughput screening assay systems in microscale fluidic devices
US5942056A (en) 1993-04-22 1999-08-24 Federalloy, Inc. Plumbing fixtures and fittings employing copper-bismuth casting alloys
WO1999042539A1 (en) 1998-02-17 1999-08-26 Sofitech N.V. Anti-accretion additives for drilling fluids
WO1999054730A1 (en) 1998-04-20 1999-10-28 Wallac Oy Method and device for carrying out a chemical analysis in small amounts of liquid
US5980936A (en) 1997-08-07 1999-11-09 Alliance Pharmaceutical Corp. Multiple emulsions comprising a hydrophobic continuous phase
US5989892A (en) 1995-06-14 1999-11-23 Tonen Corporation Microorganisms, demulsifiers and processes for breaking an emulsion
US5989815A (en) 1994-03-18 1999-11-23 University Of Utah Research Foundation Methods for detecting predisposition to cancer at the MTS gene
US5995341A (en) 1994-09-30 1999-11-30 Kabushiki Kaisha Toshiba Magnetic disk drive recording a signal with a skew angle
WO1999061888A2 (en) 1998-05-22 1999-12-02 California Institute Of Technology Microfabricated cell sorter
US5997636A (en) 1998-05-01 1999-12-07 Instrumentation Technology Associates, Inc. Method and apparatus for growing crystals
WO1999063260A1 (en) 1998-06-04 1999-12-09 The Regents Of The University Of California Microfluidic interconnects
US6008003A (en) 1997-10-28 1999-12-28 Promega Corporation Non-invasive diagnostic method for interstitial cystitis and bladder cancer
WO2000004139A1 (en) 1998-07-17 2000-01-27 Mirus Corporation Micellar systems
US6023540A (en) 1997-03-14 2000-02-08 Trustees Of Tufts College Fiber optic sensor with encoded microspheres
US6028066A (en) 1997-05-06 2000-02-22 Imarx Pharmaceutical Corp. Prodrugs comprising fluorinated amphiphiles
US6045755A (en) 1997-03-10 2000-04-04 Trega Biosciences,, Inc. Apparatus and method for combinatorial chemistry synthesis
US6048551A (en) 1997-03-27 2000-04-11 Hilfinger; John M. Microsphere encapsulation of gene transfer vectors
US6048690A (en) 1991-11-07 2000-04-11 Nanogen, Inc. Methods for electronic fluorescent perturbation for analysis and electronic perturbation catalysis for synthesis
US6074879A (en) 1997-06-23 2000-06-13 Bayer Corporation Synthetic polymer particles for use as standards and calibrators in flow cytometry
US6081612A (en) 1997-02-28 2000-06-27 Electro Optical Sciences Inc. Systems and methods for the multispectral imaging and characterization of skin tissue
WO2000037924A1 (en) 1998-12-22 2000-06-29 Research Foundation Of Cuny Time-resolved optical backscattering tomographic image reconstruction in scattering turbid media
US6086740A (en) 1998-10-29 2000-07-11 Caliper Technologies Corp. Multiplexed microfluidic devices and systems
WO2000040712A1 (en) 1999-01-07 2000-07-13 Medical Research Council Optical sorting method
US6103537A (en) 1997-10-02 2000-08-15 Aclara Biosciences, Inc. Capillary assays involving separation of free and bound species
WO2000047322A2 (en) 1999-02-12 2000-08-17 Board Of Regents, The University Of Texas System Method and apparatus for programmable fluidic processing
US6105877A (en) 1992-12-01 2000-08-22 Electrosols Ltd. Dispensing device
US6107059A (en) 1992-04-29 2000-08-22 Affymax Technologies N.V. Peptide library and screening method
US6105571A (en) 1992-12-22 2000-08-22 Electrosols, Ltd. Dispensing device
WO2000052455A1 (en) 1999-03-02 2000-09-08 Advion Biosciences, Inc. Integrated monolithic microfabricated dispensing nozzle and liquid chromatography-electrospray system and method
US6116516A (en) 1996-05-13 2000-09-12 Universidad De Sevilla Stabilized capillary microjet and devices and methods for producing same
US6118849A (en) 1997-06-27 2000-09-12 Japan Science And Technology Corporation Microstrip gas chamber high-speed data acquisition system and method of measuring samples by use of the system
US6119953A (en) 1996-05-13 2000-09-19 Aradigm Corporation Liquid atomization process
US6120666A (en) 1996-09-26 2000-09-19 Ut-Battelle, Llc Microfabricated device and method for multiplexed electrokinetic focusing of fluid streams and a transport cytometry method using same
WO2000054735A1 (en) 1999-03-17 2000-09-21 Merck Patent Gmbh Method for producing cosmetic or pharmaceutical formulations by means of a micromixture directly before use
US6124388A (en) 1995-07-19 2000-09-26 Nippon Telegraph And Telephone Corporation Water repellent composition, fluorocarbon polymer coating composition and coating film therefrom
US6124439A (en) 1994-08-17 2000-09-26 The Rockefeller University OB polypeptide antibodies and method of making
JP2000271475A (en) 1999-03-23 2000-10-03 Shinji Katsura Finely controlling method of chemical reaction by fine operation of water-in-oil emulsion
US6130098A (en) 1995-09-15 2000-10-10 The Regents Of The University Of Michigan Moving microdroplets
US6130052A (en) 1997-04-25 2000-10-10 Ludwig Institute For Cancer Research Leukemia associated genes
WO2000061275A2 (en) 1999-04-08 2000-10-19 Bernd Penth Method and device for carrying out chemical and physical processes
US6137214A (en) 1998-02-23 2000-10-24 Micron Technology, Inc. Display device with silicon-containing adhesion layer
US6139303A (en) 1998-11-20 2000-10-31 United Technologies Corporation Fixture for disposing a laser blocking material in an airfoil
US6140053A (en) 1996-11-06 2000-10-31 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US6143496A (en) 1997-04-17 2000-11-07 Cytonix Corporation Method of sampling, amplifying and quantifying segment of nucleic acid, polymerase chain reaction assembly having nanoliter-sized sample chambers, and method of filling assembly
US6146828A (en) 1996-08-14 2000-11-14 Exact Laboratories, Inc. Methods for detecting differences in RNA expression levels and uses therefor
US6149789A (en) 1990-10-31 2000-11-21 Fraunhofer Gesellschaft Zur Forderung Der Angewandten Forschung E.V. Process for manipulating microscopic, dielectric particles and a device therefor
WO2000070080A1 (en) 1999-05-17 2000-11-23 Caliper Technologies Corp. Focusing of microparticles in microfluidic systems
US6155710A (en) 1996-02-20 2000-12-05 Japan, As Represented By Director Of National Food Research Institute, Ministry Of Agriculture, Forestry And Fisheries Method and device for producing emulsions
US6162421A (en) 1997-11-17 2000-12-19 Revlon Consumer Products Corporation Pigmented water-in-oil emulsion cosmetic sticks
WO2000076673A1 (en) 1999-06-11 2000-12-21 Aradigm Corporation Method for producing an aerosol
US6165778A (en) 1993-11-02 2000-12-26 Affymax Technologies N.V. Reaction vessel agitation apparatus
WO2000078455A1 (en) 1999-06-22 2000-12-28 Tecan Trading Ag Devices and methods for the performance of miniaturized in vitro amplification assays
US6171796B1 (en) 1998-01-21 2001-01-09 Urocor, Inc. Biomarkers and targets for diagnosis prognosis and management of prostate disease
US6171850B1 (en) 1999-03-08 2001-01-09 Caliper Technologies Corp. Integrated devices and systems for performing temperature controlled reactions and analyses
US6174160B1 (en) 1999-03-25 2001-01-16 University Of Washington Staged prevaporizer-premixer
US6177479B1 (en) 1998-03-30 2001-01-23 Japan As Represented By Director Of National Food Research Institute, Ministry Of Agriculture, Forestry And Fisheries Continuous manufacturing method for microspheres and apparatus
US6180372B1 (en) 1997-04-23 2001-01-30 Bruker Daltonik Gmbh Method and devices for extremely fast DNA replication by polymerase chain reactions (PCR)
WO2001009598A1 (en) 1999-07-28 2001-02-08 University Of Washington Fluidic interconnect, interconnect manifold and microfluidic devices for internal delivery of gases and application of vacuum
US6187214B1 (en) 1996-05-13 2001-02-13 Universidad De Seville Method and device for production of components for microfabrication
US6189803B1 (en) 1996-05-13 2001-02-20 University Of Seville Fuel injection nozzle and method of use
WO2001012327A1 (en) 1999-08-12 2001-02-22 Ut-Battelle, Llc Microfluidic devices for the controlled manipulation of small volumes
WO2001014589A2 (en) 1999-08-20 2001-03-01 Luminex Corporation Liquid array technology
US6196525B1 (en) 1996-05-13 2001-03-06 Universidad De Sevilla Device and method for fluid aeration via gas forced through a liquid within an orifice of a pressure chamber
US6197335B1 (en) 1997-09-16 2001-03-06 Bernard Charles Sherman Solid pharmaceutical compositions comprising a cyclosporin and an anionic surfactant
WO2001018244A2 (en) 1999-09-08 2001-03-15 Medical Research Council Selection system
US6203993B1 (en) 1996-08-14 2001-03-20 Exact Science Corp. Methods for the detection of nucleic acids
US6208749B1 (en) 1997-02-28 2001-03-27 Electro-Optical Sciences, Inc. Systems and methods for the multispectral imaging and characterization of skin tissue
US6207397B1 (en) 1996-04-18 2001-03-27 Ariad Pharmaceuticals, Inc. In vitro fluorescence polarization assay
US6210396B1 (en) 1999-06-24 2001-04-03 Medtronic, Inc. Guiding catheter with tungsten loaded band
US6210896B1 (en) 1998-08-13 2001-04-03 Us Genomics Molecular motors
US6210891B1 (en) 1996-09-27 2001-04-03 Pyrosequencing Ab Method of sequencing DNA
US6221654B1 (en) 1996-09-25 2001-04-24 California Institute Of Technology Method and apparatus for analysis and sorting of polynucleotides based on size
US6227466B1 (en) 1998-08-04 2001-05-08 William J. Hartman Electrostatic spray module
US6235383B1 (en) 1997-01-24 2001-05-22 Samsung Corning Co., Ltd. Glass article having a durable water repellent surface
US6243373B1 (en) 1995-11-01 2001-06-05 Telecom Internet Ltd. Method and apparatus for implementing a computer network/internet telephone system
US6248378B1 (en) 1998-12-16 2001-06-19 Universidad De Sevilla Enhanced food products
US6252129B1 (en) 1996-07-23 2001-06-26 Electrosols, Ltd. Dispensing device and method for forming material
US6251661B1 (en) 1997-05-14 2001-06-26 Morishita Jintan Co., Ltd. Seamless capsule for synthesizing biopolymer and method for producing the same
DE19961257A1 (en) 1999-12-18 2001-07-05 Inst Mikrotechnik Mainz Gmbh Micromixer
US6258568B1 (en) 1996-12-23 2001-07-10 Pyrosequencing Ab Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation
US6258858B1 (en) 1998-07-02 2001-07-10 Japan As Represented By Director Of National Food Research Institute, Ministry Of Agriculture, Forestry And Fisheries Cross-flow microchannel apparatus and method of producing or separating emulsions making use thereof
US6263222B1 (en) 1991-03-07 2001-07-17 Masimo Corporation Signal processing apparatus
US6261661B1 (en) 1997-07-23 2001-07-17 Matsushita Electrical Industrial Co., Ltd. Optical information recording medium and method for producing the same
US6261797B1 (en) 1996-01-29 2001-07-17 Stratagene Primer-mediated polynucleotide synthesis and manipulation techniques
US6268222B1 (en) 1998-01-22 2001-07-31 Luminex Corporation Microparticles attached to nanoparticles labeled with flourescent dye
US6267353B1 (en) 1999-04-19 2001-07-31 Pbm, Inc. Self draining valve
US6268165B1 (en) 1997-03-19 2001-07-31 The Board Of Trustees Of The University Of Arkansas Methods for the early diagnosis of ovarian cancer
US6268152B1 (en) 1993-06-25 2001-07-31 Affymetrix, Inc. Probe kit for identifying a base in a nucleic acid
US6274320B1 (en) 1999-09-16 2001-08-14 Curagen Corporation Method of sequencing a nucleic acid
US6280948B1 (en) 1997-03-11 2001-08-28 Wisconsin Alumni Research Foundation Nucleic acid indexing
WO2001064332A1 (en) 2000-03-02 2001-09-07 Newcastle Universtiy Ventures Limited Capillary reactor distribution device and method
US6292756B1 (en) 1998-02-26 2001-09-18 Premier Instruments, Inc. Narrow band infrared water fraction apparatus for gas well and liquid hydrocarbon flow stream use
WO2001069289A2 (en) 2000-03-10 2001-09-20 Flow Focusing, Inc. Methods for producing optical fiber by focusing high viscosity liquid
US20010023078A1 (en) 1998-09-18 2001-09-20 Massachusetts Institute Of Technology Biological applications of quantum dots
WO2001068257A1 (en) 2000-03-10 2001-09-20 Bioprocessors Corporation Microreactor
US6294344B1 (en) 1997-03-19 2001-09-25 The Board Of Trustees Of The University Of Arkansas Methods for the early diagnosis of ovarian cancer
US20010024790A1 (en) 2000-03-17 2001-09-27 Hitachi, Ltd. DNA base sequencing system
US6296020B1 (en) 1998-10-13 2001-10-02 Biomicro Systems, Inc. Fluid circuit components based upon passive fluid dynamics
US6296673B1 (en) 1999-06-18 2001-10-02 The Regents Of The University Of California Methods and apparatus for performing array microcrystallizations
WO2001072431A1 (en) 2000-03-28 2001-10-04 Nisco Engineering Ag Method and device for producing drops of equal size
US6301055B1 (en) 2000-08-16 2001-10-09 California Institute Of Technology Solid immersion lens structures and methods for producing solid immersion lens structures
US6299145B1 (en) 1996-05-13 2001-10-09 Universidad De Sevilla Device and method for fluid aeration via gas forced through a liquid within an orifice of a pressure chamber
US20010032053A1 (en) 2000-01-24 2001-10-18 Hielscher Andreas H. Imaging of a scattering medium using the equation of radiative transfer
US20010029983A1 (en) 1999-06-28 2001-10-18 Unger Marc A. Microfabricated elastomeric valve and pump systems
US6307957B1 (en) 1997-02-28 2001-10-23 Electro-Optical Sciences Inc Multispectral imaging and characterization of biological tissue
US20010034025A1 (en) 1999-01-15 2001-10-25 Ljl Biosystems, Inc. Methods and apparatus for detecting polynucleotide hybridization
WO2001080283A1 (en) 2000-04-18 2001-10-25 Waters Investments Limited Improved electrospray and other lc/ms interfaces
US6309842B1 (en) 1996-12-03 2001-10-30 Glaxo Wellcome Inc. Use of modified tethers in screening compound libraries
US6310653B1 (en) 1995-12-12 2001-10-30 Ronald D. Malcolm, Jr. Phase comparison and phase adjustment for synchronization to a reference signal that is asynchronous with respect to a digital sampling clock
JP2001301154A (en) 2000-04-20 2001-10-30 Dainippon Printing Co Ltd Method of attaching liquid with surface tension reduced by voltage application by electric field jet
US6310354B1 (en) 1996-12-03 2001-10-30 Erkki Soini Method and a device for monitoring nucleic acid amplification reactions
US6316208B1 (en) 1994-01-07 2001-11-13 Memorial Sloan-Kettering Cancer Center Methods for determining isolated p27 protein levels and uses thereof
US6316213B1 (en) 1997-03-19 2001-11-13 The Board Of Trustees Of The University Of Arkansas Methods for the early diagnosis of ovarian, breast and lung cancer
US20010041344A1 (en) 2000-02-03 2001-11-15 Nanoscale Combinatorial Synthesis, Inc., 625 Clyde Avenue, Mountain View, Ca 94043 Nonredundant split/pool synthesis of combinatorial libraries
US20010041357A1 (en) 1999-07-28 2001-11-15 Yves Fouillet Method for carrying out a biochemical protocol in continuous flow in a microreactor
US20010041343A1 (en) 1999-05-04 2001-11-15 Pankowsky Dan A. Products and methods for single parameter and multiparameter phenotyping of cells
US6324417B1 (en) 1996-11-19 2001-11-27 Optiscan Limited Method for measurement of skin histology
WO2001089788A2 (en) 2000-05-25 2001-11-29 President And Fellows Of Harvard College Patterning of surfaces utilizing microfluidic stamps including three-dimensionally arrayed channel networks
WO2001089787A2 (en) 2000-05-25 2001-11-29 President And Fellows Of Harvard College Microfluidic systems including three-dimensionally arrayed channel networks
US6326145B1 (en) 1998-06-13 2001-12-04 Zeneca Limited Methods for detecting target nucleic acid sequences
US20010048900A1 (en) 2000-05-24 2001-12-06 Bardell Ronald L. Jet vortex mixer
WO2001094635A2 (en) 2000-06-05 2001-12-13 California Institute Of Technology Integrated active flux microfluidic devices and methods
US20010050881A1 (en) 1999-09-20 2001-12-13 Depaoli David W. Continuous flow, electrohydrodynamic micromixing apparatus and methods
US6336463B1 (en) 1998-03-31 2002-01-08 Nec Corporation Cleaning/drying station and production line for semiconductor devices
US20020004532A1 (en) 2000-05-26 2002-01-10 Michelle Matathia Low emulsifier multiple emulsions
US20020005354A1 (en) 1997-09-23 2002-01-17 California Institute Of Technology Microfabricated cell sorter
US20020008028A1 (en) 2000-01-12 2002-01-24 Jacobson Stephen C. Microfluidic device and method for focusing, segmenting, and dispensing of a fluid stream
US20020012971A1 (en) 2000-03-20 2002-01-31 Mehta Tammy Burd PCR compatible nucleic acid sieving medium
US20020015997A1 (en) 1997-06-16 2002-02-07 Lafferty William Michael Capillary array-based sample screening
US20020022038A1 (en) 2000-05-05 2002-02-21 Bruno Biatry Microcapsules with an aqueous core containing at least one water-soluble cosmetic or dermatological active principle and cosmetic or dermatological compositions containing them
US20020022261A1 (en) 1995-06-29 2002-02-21 Anderson Rolfe C. Miniaturized genetic analysis systems and methods
WO2002016017A2 (en) 2000-08-25 2002-02-28 INSTITUT FüR MIKROTECHNIK MAINZ GMBH Method and statistical micromixer for mixing at least two liquids
WO2002018949A2 (en) 2000-08-31 2002-03-07 The Regents Of The University Of California Capillary array and related methods
US6355198B1 (en) 1996-03-15 2002-03-12 President And Fellows Of Harvard College Method of forming articles including waveguides via capillary micromolding and microtransfer molding
US6355193B1 (en) 2000-03-01 2002-03-12 Gale Stott Method for making a faux stone concrete panel
WO2002023163A1 (en) 2000-09-15 2002-03-21 California Institute Of Technology Microfabricated crossflow devices and methods
WO2002022869A2 (en) 2000-09-13 2002-03-21 Medical Research Council Directed evolution method
US20020034737A1 (en) 1997-03-04 2002-03-21 Hyseq, Inc. Methods and compositions for detection or quantification of nucleic acid species
US20020033422A1 (en) 1996-05-13 2002-03-21 Ganan-Calvo Alfonso M. Fuel injection nozzle and method of use
JP2002085961A (en) 2000-09-13 2002-03-26 Inst Of Physical & Chemical Res REACTOR AND ITS MANUFACTURING METHOD
US20020036018A1 (en) 1998-10-13 2002-03-28 Mcneely Michael R. Fluid circuit components based upon passive fluid dynamics
WO2002027660A2 (en) 2000-09-29 2002-04-04 Koninklijke Philips Electronics N.V. Image registration system and method using cross-entropy optimization
US20020041378A1 (en) 2000-08-08 2002-04-11 International Business Machines Corporation Polarized light fluorescence imageing device
WO2002031203A2 (en) 2000-10-10 2002-04-18 Diversa Corporation High throughput or capillary-based screening for a bioactivity or biomolecule
WO2002036815A2 (en) 2000-10-28 2002-05-10 Ncimb Ltd. Genetic analysis of microorganisms
AU747464B2 (en) 1998-06-08 2002-05-16 Caliper Technologies Corporation Microfluidic devices, systems and methods for performing integrated reactions and separations
US20020065609A1 (en) 2000-04-10 2002-05-30 Matthew Ashby Methods for the survey and genetic analysis of populations
US6399339B1 (en) 1998-12-14 2002-06-04 Forschungszentrum Julich Gmbh Method for the enantioselective reduction of 3,5-dioxocarboxylic acids, their salts and their esters
US20020067800A1 (en) 2000-10-19 2002-06-06 Janet Newman Apparatus and method for identification of crystals by in-situ X-ray diffraction
US6403373B1 (en) 1997-10-10 2002-06-11 Ludwig Institute For Cancer Research Isolated nucleic acid molecules associated with colon, renal, and stomach cancer and methods of using these
US6405936B1 (en) 1996-05-13 2002-06-18 Universidad De Sevilla Stabilized capillary microjet and devices and methods for producing same
WO2002047665A2 (en) 2000-12-07 2002-06-20 President And Fellows Of Harvard College Methods and compositions for encapsulating active agents
US6409832B2 (en) 2000-03-31 2002-06-25 Micronics, Inc. Protein crystallization in microfluidic structures
US20020085961A1 (en) 2000-09-15 2002-07-04 Morin Jean X. Procedure and apparatus for the cleaning of flue gases containing sulfur dioxide
US20020084417A1 (en) 1998-11-23 2002-07-04 Khalil Omar S. Non-invasive sensor capable of determining optical parameters in a sample having multiple layers
US20020090720A1 (en) 2000-11-29 2002-07-11 Mutz Mitchell W. Focused acoustic ejection cell sorting system and method
US6429148B1 (en) 2001-10-09 2002-08-06 Promos Technologies, Inc. Anisotropic formation process of oxide layers for vertical transistors
US6428053B1 (en) * 1999-03-12 2002-08-06 California Institute Of Technology Micromachined fluidic coupler and method of making the same
US20020106667A1 (en) 2000-08-31 2002-08-08 Nobuko Yamamoto Screening method for gene variation
WO2002060275A1 (en) 2001-01-31 2002-08-08 Kraft Foods Holdings, Inc. Production of capsules and particles for improvement of food products
WO2002060591A1 (en) 2001-01-31 2002-08-08 Universidad De Sevilla Device and method for producing stationary multi-component liquid capillary streams and micrometric and nanometric sized capsules
US6432143B2 (en) 1997-05-16 2002-08-13 Life Technologies, Inc. Automated liquid manufacturing system
US6439103B1 (en) 1999-09-07 2002-08-27 Vector Engineering Co. Hydraulic and pneumatic cylinder construction
US6440706B1 (en) 1999-08-02 2002-08-27 Johns Hopkins University Digital amplification
US6440760B1 (en) 1999-09-18 2002-08-27 Samsung Electronics Co., Ltd. Method of measuring etched state of semiconductor wafer using optical impedence measurement
US20020119455A1 (en) 1997-02-12 2002-08-29 Chan Eugene Y. Methods and products for analyzing polymers
WO2002066992A1 (en) 2001-02-23 2002-08-29 Japan Science And Technology Corporation Small liquid particle handling method, and device therefor
WO2002068104A1 (en) 2001-02-23 2002-09-06 Japan Science And Technology Corporation Process for producing emulsion and microcapsules and apparatus therefor
US20020127591A1 (en) 2000-01-06 2002-09-12 Caliper Technologies Corp. Methods and systems for monitoring intracellular binding reactions
US6450139B1 (en) 2000-10-20 2002-09-17 Unisia Jecs Corporation Valve timing control system for internal combustion engine
US6450189B1 (en) 1998-11-13 2002-09-17 Universidad De Sevilla Method and device for production of components for microfabrication
US6454193B1 (en) 1999-04-23 2002-09-24 Battellepharma, Inc. High mass transfer electrosprayer
US20020142344A1 (en) 1998-12-11 2002-10-03 Mark Akeson Targeted molecular bar codes and methods for using the same
US20020143437A1 (en) 2001-03-28 2002-10-03 Kalyan Handique Methods and systems for control of microfluidic devices
US6464336B1 (en) 2001-10-31 2002-10-15 Eastman Kodak Company Ink jet printing with color-balanced ink drops mixed using bleached ink
US6469094B1 (en) 2000-04-28 2002-10-22 Xerox Corporation Polymerization processes
US20020155080A1 (en) 2001-03-05 2002-10-24 Glenn Robert Wayne Delivery of reactive agents via multiple emulsions for use in shelf stable products
US20020158027A1 (en) 1998-09-17 2002-10-31 Moon James E. Integrated monolithic microfabricated electrospray and liquid chromatography system and method
US6475441B1 (en) 1997-06-09 2002-11-05 Caliper Technologies Corp. Method for in situ concentration and/or dilution of materials in microfluidic systems
US20020164271A1 (en) 2001-05-02 2002-11-07 Ho Winston Z. Wavelength-coded bead for bioassay and signature recogniton
US20020164629A1 (en) 2001-03-12 2002-11-07 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences by asynchronous base extension
US20020166582A1 (en) 2000-04-14 2002-11-14 Nanostream, Inc. Microfluidic branch metering systems and methods
US6481648B1 (en) 1999-10-01 2002-11-19 Agilent Technologies, Inc. Spray tip for a microfluidic laboratory microchip
US20020179849A1 (en) 1999-05-12 2002-12-05 Kevin Maher Multiplexed fluorescent detection in microfluidic devices
WO2002103011A2 (en) 2001-06-18 2002-12-27 Medical Research Council Selective gene amplification
WO2002103363A2 (en) 2001-06-18 2002-12-27 Medical Research Council Selection by avidity capture
US6503933B1 (en) 1998-02-19 2003-01-07 Aventis Cropscience Uk Limited 2-pyridylmethylamine derivatives useful as fungicides
US20030008308A1 (en) 2001-04-06 2003-01-09 California Institute Of Technology Nucleic acid amplification utilizing microfluidic devices
WO2003003015A2 (en) 2001-06-28 2003-01-09 Advanced Research And Technology Institute, Inc. Methods of preparing multicolor quantum dot tagged beads and conjugates thereof
US20030012586A1 (en) 2001-05-24 2003-01-16 Nobuo Iwata Developer container, developing conveying device and image forming apparatus using the same
US6508988B1 (en) 2000-10-03 2003-01-21 California Institute Of Technology Combinatorial synthesis system
US20030017579A1 (en) 2001-07-10 2003-01-23 Corn Robert M. Surface plasmon resonance imaging of micro-arrays
US20030015425A1 (en) 2001-06-20 2003-01-23 Coventor Inc. Microfluidic system including a virtual wall fluid interface port for interfacing fluids with the microfluidic system
US20030017305A1 (en) 2001-07-17 2003-01-23 Roitman Daniel B. Paek embossing and adhesion for microfluidic devices
US6511803B1 (en) 1997-10-10 2003-01-28 President And Fellows Of Harvard College Replica amplification of nucleic acid arrays
WO2003011443A2 (en) 2001-07-27 2003-02-13 President And Fellows Of Harvard College Laminar mixing apparatus and methods
US6520425B1 (en) 2001-08-21 2003-02-18 The University Of Akron Process and apparatus for the production of nanofibers
US20030040620A1 (en) 2000-05-20 2003-02-27 Langmore John P. Method of producing a DNA library using positional amplification
US20030038865A1 (en) * 2001-08-22 2003-02-27 Shusuke Inamura Ink supply mechanism and inkjet recording apparatus including the ink supply mechanism
US6530944B2 (en) 2000-02-08 2003-03-11 Rice University Optically-active nanoparticles for use in therapeutic and diagnostic methods
US20030059764A1 (en) 2000-10-18 2003-03-27 Ilya Ravkin Multiplexed cell analysis system
US6540895B1 (en) 1997-09-23 2003-04-01 California Institute Of Technology Microfabricated cell sorter for chemical and biological materials
US6540395B2 (en) 1999-12-23 2003-04-01 Ernst Mühlbauer KG Dynamic mixer for dental impression compounds
US20030064414A1 (en) 2001-03-30 2003-04-03 Benecky Michael J. Rapid assessment of coagulation activity in whole blood
US20030061687A1 (en) 2000-06-27 2003-04-03 California Institute Of Technology, A California Corporation High throughput screening of crystallization materials
WO2003026798A1 (en) 2001-09-21 2003-04-03 Commissariat A L'energie Atomique Method for moving a fluid of interest in a capillary tube and fluidic microsystem
US6553960B1 (en) 1997-04-11 2003-04-29 Yanmar Co., Ltd. Combustion system for direct injection diesel engines
US6553944B1 (en) 2001-07-03 2003-04-29 Virginia A. Allen Wrist worn leash retaining device
US20030082795A1 (en) 2001-04-25 2003-05-01 Michael Shuler Devices and methods for pharmacokinetic-based cell culture system
US20030083276A1 (en) 1998-06-30 2003-05-01 Li Gloria C. Uses of DNA-PK
US6557334B2 (en) 2001-07-13 2003-05-06 Willem Jager Caster mounted reel mower
WO2003037302A1 (en) 2001-10-30 2003-05-08 Windsor J Brian Method and system for the co-isolation of cognate dna, rna and protein sequences and method for screening co-isolates for defined activities
US6565010B2 (en) 2000-03-24 2003-05-20 Praxair Technology, Inc. Hot gas atomization
JP2003149136A (en) 2001-11-13 2003-05-21 Shimadzu Corp Optical image measurement method
US6569631B1 (en) 1998-11-12 2003-05-27 3-Dimensional Pharmaceuticals, Inc. Microplate thermal shift assay for ligand development using 5-(4″dimethylaminophenyl)-2-(4′-phenyl)oxazole derivative fluorescent dyes
WO2003044187A2 (en) 2001-11-16 2003-05-30 Medical Research Council Emulsion compositions
US20030104372A1 (en) 1996-12-23 2003-06-05 Pyrosequencing Ab. Allele specific primer extension
US6576420B1 (en) 1998-06-23 2003-06-10 Regents Of The University Of California Method for early diagnosis of, and determination of prognosis in, cancer
US20030108900A1 (en) 2001-07-12 2003-06-12 Arnold Oliphant Multiplex nucleic acid reactions
US6592821B1 (en) 1999-05-17 2003-07-15 Caliper Technologies Corp. Focusing of microparticles in microfluidic systems
US6592321B2 (en) 2000-08-03 2003-07-15 Demag Cranes & Components Gmbh Control and guiding device for manually operating a handling unit, and modular construction kit for making such devices of different configuration
US6591852B1 (en) 1998-10-13 2003-07-15 Biomicro Systems, Inc. Fluid circuit components based upon passive fluid dynamics
US20030144260A1 (en) 2002-01-03 2003-07-31 Yissum Research Development Company Of The Hebrew University Of Jerusalem Heterocyclic compounds, method of developing new drug leads and combinatorial libraries used in such method
US20030143599A1 (en) 2001-11-13 2003-07-31 Rubicon Genomics Inc. DNA amplification and sequencing using DNA molecules generated by random fragmentation
US20030148273A1 (en) 2000-08-26 2003-08-07 Shoulian Dong Target enrichment and amplification
JP2003222633A (en) 2002-01-30 2003-08-08 Nippon Sheet Glass Co Ltd Microchip
JP2003232525A (en) 2002-02-08 2003-08-22 Mitsubishi Electric Corp Induction heating cooker
US6614598B1 (en) 1998-11-12 2003-09-02 Institute Of Technology, California Microlensing particles and applications
WO2003078659A2 (en) 2002-03-20 2003-09-25 Innovativebio.Biz Microcapsules with controlable permeability encapsulating a nucleic acid amplification reaction mixture and their use as reaction compartments for parallels reactions
US20030181574A1 (en) 2002-02-11 2003-09-25 Herve Adam Method for controlling the stability of emulsions
US6627603B1 (en) 1997-08-07 2003-09-30 Centre National De La Recherche Scientifiquue (C.N.R.S.) Method for releasing an active principle contained a multiple emulsion
US20030183525A1 (en) 2002-04-01 2003-10-02 Xerox Corporation Apparatus and method for using electrostatic force to cause fluid movement
US6630006B2 (en) 1999-06-18 2003-10-07 The Regents Of The University Of California Method for screening microcrystallizations for crystal formation
US6632619B1 (en) 1997-05-16 2003-10-14 The Governors Of The University Of Alberta Microfluidic system and methods of use
US6638749B1 (en) 1995-11-13 2003-10-28 Genencor International, Inc. Carbon dioxide soluble surfactant having two fluoroether CO2-philic tail groups and a head group
US6637463B1 (en) 1998-10-13 2003-10-28 Biomicro Systems, Inc. Multi-channel microfluidic system design with balanced fluid flow distribution
US20030207295A1 (en) 1999-04-20 2003-11-06 Kevin Gunderson Detection of nucleic acid reactions on bead arrays
US6646253B1 (en) 1998-05-20 2003-11-11 GSF-Forschungszentrum für Umwelt und Gesundheit GmbH Gas inlet for an ion source
US6653626B2 (en) 1994-07-11 2003-11-25 Agilent Technologies, Inc. Ion sampling for APPI mass spectrometry
US20030219754A1 (en) 2002-05-23 2003-11-27 Oleksy Jerome E. Fluorescence polarization detection of nucleic acids
US6656267B2 (en) 2001-07-10 2003-12-02 Structural Genomix, Inc. Tray for macromolecule crystallization and method of using the same
US20030224509A1 (en) 2000-11-29 2003-12-04 Cangen International DAP-kinase and HOXA9, two human genes associated with genesis, progression, and aggressiveness of non-small cell lung cancer
WO2003099843A2 (en) 2002-05-20 2003-12-04 Dow Corning Corporation Peptide derivatives, and their use for the synthesis of silicon-based composite materials
US6659370B1 (en) 1998-05-25 2003-12-09 Fuji Bc Engineering Co., Ltd. Liquid spray device and cutting method
US20030229376A1 (en) 1999-06-01 2003-12-11 Biointeractions Ltd. Coated surfaces for immobilizing negatively charged anticoagulating agents from blood fluid
US20030230486A1 (en) 2002-03-05 2003-12-18 Caliper Technologies Corp. Mixed mode microfluidic systems
US20030232356A1 (en) 2002-02-08 2003-12-18 Dooley Thomas P. Skin cell biomarkers and methods for identifying biomarkers using nucleic acid microarrays
US6670142B2 (en) 2001-10-26 2003-12-30 The Regents Of The University Of California Method for screening combinatorial bead library, capturing cells from body fluids, and ligands for cancer cells
WO2004002627A2 (en) 2002-06-28 2004-01-08 President And Fellows Of Harvard College Method and apparatus for fluid dispersion
US20040005582A1 (en) 2000-08-10 2004-01-08 Nanobiodynamics, Incorporated Biospecific desorption microflow systems and methods for studying biospecific interactions and their modulators
US6679441B1 (en) 1998-03-27 2004-01-20 Centre National De La Recherche Scientifique (C.N.R.S.) Electrohydrodynamic spraying means
US6680178B2 (en) 2000-06-02 2004-01-20 The Regents Of The University Of California Profiling of protease specificity using combinatorial fluorogenic substrate libraries
US6682890B2 (en) 2000-08-17 2004-01-27 Protein Design Labs, Inc. Methods of diagnosing and determining prognosis of colorectal cancer
US20040018525A1 (en) 2002-05-21 2004-01-29 Bayer Aktiengesellschaft Methods and compositions for the prediction, diagnosis, prognosis, prevention and treatment of malignant neoplasma
US20040030255A1 (en) 2002-06-05 2004-02-12 Research Foundation Of City University Hybrid-dual-Fourier tomographic algorithm for a fast three-dimensional optical image reconstruction in turbid media
US20040031688A1 (en) 1999-01-25 2004-02-19 Shenderov Alexander David Actuators for microfluidics without moving parts
US20040037739A1 (en) 2001-03-09 2004-02-26 Mcneely Michael Method and system for microfluidic interfacing to arrays
US20040037813A1 (en) 1999-02-25 2004-02-26 Simpson David G. Electroprocessed collagen and tissue engineering
WO2004018497A2 (en) 2002-08-23 2004-03-04 Solexa Limited Modified nucleotides for polynucleotide sequencing
US20040050946A1 (en) 2002-08-06 2004-03-18 Clean Earth Technologies, Llc Method and apparatus for electrostatic spray
US20040053247A1 (en) 1997-12-01 2004-03-18 Sloan-Kettering Institute For Cancer Research Markers for prostate cancer
US20040057906A1 (en) 2002-09-24 2004-03-25 Li-Chien Hsu Perfluorocarbon emulsions with non-fluorinated surfactants
WO2004024917A2 (en) 2002-09-11 2004-03-25 Medical Research Council Single-molecule in vitro evolution
US20040058450A1 (en) 2002-09-24 2004-03-25 Pamula Vamsee K. Methods and apparatus for manipulating droplets by electrowetting-based techniques
WO2004026453A2 (en) 2002-09-06 2004-04-01 Genteric, Inc. Microcapsules and methods of use
US6717136B2 (en) 2001-03-19 2004-04-06 Gyros Ab Microfludic system (EDI)
US20040071781A1 (en) 2002-10-11 2004-04-15 Ferro Corporation Composite particles and method for preparing
US20040079881A1 (en) 2002-09-18 2004-04-29 Fischer Steven M. Multimode ionization source
US6729561B2 (en) 2000-05-30 2004-05-04 Dainippon Screen Mfg. Co., Ltd. Cleaning nozzle and substrate cleaning apparatus
WO2004038363A2 (en) 2002-05-09 2004-05-06 The University Of Chicago Microfluidic device and method for pressure-driven plug transport and reaction
WO2004037374A2 (en) 2002-10-23 2004-05-06 The Trustees Of Princeton University Method for continuous particle separation using obstacle arrays asymmetrically aligned to fields
US20040086892A1 (en) 2002-11-06 2004-05-06 Crothers Donald M. Universal tag assay
US20040091923A1 (en) 1993-07-23 2004-05-13 Bio-Rad Laboratories, Inc. Linked linear amplification of nucleic acids
US20040092824A1 (en) 2001-03-06 2004-05-13 Stamnes Jakob J. Method and an arrangement for the determination of the optical properties of a multi-layered tissue
US6738502B1 (en) 1999-06-04 2004-05-18 Kairos Scientific, Inc. Multispectral taxonomic identification
US20040096515A1 (en) 2001-12-07 2004-05-20 Bausch Andreas R. Methods and compositions for encapsulating active agents
US6739036B2 (en) 2000-09-13 2004-05-25 Fuji Machine Mfg., Co., Ltd. Electric-component mounting system
US20040101822A1 (en) 2002-11-26 2004-05-27 Ulrich Wiesner Fluorescent silica-based nanoparticles
US6744046B2 (en) 2001-05-24 2004-06-01 New Objective, Inc. Method and apparatus for feedback controlled electrospray
US6752922B2 (en) 2001-04-06 2004-06-22 Fluidigm Corporation Microfluidic chromatography
US20040136497A1 (en) 2002-10-30 2004-07-15 Meldrum Deirdre R Preparation of samples and sample evaluation
US20040142329A1 (en) 2003-01-17 2004-07-22 Ingeneus Corporation Probe conjugation to increase multiplex binding motif preference
US6767704B2 (en) 2000-03-27 2004-07-27 Thomas Jefferson University Methods of screening and diagnosing esophageal cancer by determining guanylin cyclase C expression
US6766817B2 (en) 2001-07-25 2004-07-27 Tubarc Technologies, Llc Fluid conduction utilizing a reversible unsaturated siphon with tubarc porosity action
US6767194B2 (en) 2001-01-08 2004-07-27 President And Fellows Of Harvard College Valves and pumps for microfluidic systems and method for making microfluidic systems
US20040146921A1 (en) 2003-01-24 2004-07-29 Bayer Pharmaceuticals Corporation Expression profiles for colon cancer and methods of use
US20040146866A1 (en) 2003-01-21 2004-07-29 Guoliang Fu Quantitative multiplex detection of nucleic acids
US20040159633A1 (en) 1993-10-04 2004-08-19 President & Fellows Of Harvard University Methods of etching articles via micro contact printing
WO2004070005A2 (en) 2003-01-29 2004-08-19 454 Corporation Double ended sequencing
WO2004071638A2 (en) 2003-02-11 2004-08-26 Regents Of The University Of California, The Microfluidic devices and method for controlled viscous shearing and formation of amphiphilic vesicles
US20040181343A1 (en) 2002-11-01 2004-09-16 Cellectricon Ab Computer program products and systems for rapidly changing the solution environment around sensors
US20040180346A1 (en) 2003-03-14 2004-09-16 The Regents Of The University Of California. Chemical amplification based on fluid partitioning
US20040181131A1 (en) 2003-02-25 2004-09-16 Maynard John D. Determination of pH including hemoglobin correction
US20040182712A1 (en) 2003-03-20 2004-09-23 Basol Bulent M. Process and system for eliminating gas bubbles during electrochemical processing
US6797056B2 (en) 2001-06-08 2004-09-28 Syrrx, Inc. Microfluidic method employing delivery of plural different fluids to same lumen
EP1462517A1 (en) 2002-01-08 2004-09-29 Japan Science and Technology Agency Pcr method by electrostatic transportation, hybridization method for electrostatic transportation and devices therefor
US20040188254A1 (en) 2002-03-05 2004-09-30 Caliper Technologies Corp. Mixed mode microfluidic systems
WO2004083443A1 (en) 2002-12-20 2004-09-30 Caliper Life Sciences, Inc. Single molecule amplification and detection of dna
US6800849B2 (en) 2001-12-19 2004-10-05 Sau Lan Tang Staats Microfluidic array devices and methods of manufacture and uses thereof
CA2520548A1 (en) 2003-03-31 2004-10-14 Medical Research Council Selection by compartmentalised screening
WO2004087308A1 (en) 2003-03-31 2004-10-14 Medical Research Council Method of synthesis and testing of combinatorial libraries using microcapsules
US6806058B2 (en) 2001-05-26 2004-10-19 One Cell Systems, Inc. Secretions of proteins by encapsulated cells
US20040209299A1 (en) 2003-03-07 2004-10-21 Rubicon Genomics, Inc. In vitro DNA immortalization and whole genome amplification using libraries generated from randomly fragmented DNA
US6808382B2 (en) 2000-03-15 2004-10-26 Lino Lanfranchi Device for conveying and checking containers, in particular preforms
WO2004091763A2 (en) 2003-04-10 2004-10-28 President And Fellows Of Harvard College Formation and control of fluidic species
US6814980B2 (en) 1998-04-23 2004-11-09 The Regents Of The University Of Michigan Microspheres containing condensed polyanionic bioactive agents and methods for their production
US20040224419A1 (en) 2003-05-06 2004-11-11 Thrombodyne, Inc. Systems and methods for measuring fluid properties
US6818395B1 (en) 1999-06-28 2004-11-16 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences
US20040229349A1 (en) 2002-04-01 2004-11-18 Fluidigm Corporation Microfluidic particle-analysis systems
WO2004102204A1 (en) 2003-05-16 2004-11-25 Global Technologies (Nz) Ltd Method and apparatus for mixing sample and reagent in a suspension fluid
WO2004103565A2 (en) 2003-05-19 2004-12-02 Hans-Knöll-Institut für Naturstoff-Forschung e.V. Device and method for structuring liquids and for dosing reaction liquids into liquid compartments immersed in a separation medium
DE10322893A1 (en) 2003-05-19 2004-12-16 Hans-Knöll-Institut für Naturstoff-Forschung e.V. Equipment for microtechnological structuring of fluids used in analytical or combinatorial biology or chemistry, has dosing, splitting and fusion devices in fluid pathway
US6833242B2 (en) 1997-09-23 2004-12-21 California Institute Of Technology Methods for detecting and sorting polynucleotides based on size
US6832787B1 (en) 2003-01-24 2004-12-21 Sandia National Laboratories Edge compression manifold apparatus
US20040258203A1 (en) 2002-06-17 2004-12-23 Akihito Yamano Crystal evaluating device
US20040259083A1 (en) 2001-05-11 2004-12-23 Mitsuaki Oshima Biomolecular substrate and method and apparatus for examination and diagnosis using the same
US20050000970A1 (en) 2003-03-25 2005-01-06 Toyota Jidosha Kabushiki Kaisha Gas storage tank and method of manufacturing the same
WO2005000970A1 (en) 2003-06-30 2005-01-06 Raustech Pty Ltd Charged emulsions for site-specific deposition of matter at micro and nano scale
US6841350B2 (en) 1999-02-20 2005-01-11 The North West London Hospitals Nhs Trust Of Northwick Park Hospital Methods of diagnosing prostate cancer through the detection of the presence or absence of Pax 2 mRNA
US20050008592A1 (en) 2002-06-26 2005-01-13 Nadia Gardel Water-in-oil emulsion foundation
WO2005002730A1 (en) 2003-07-02 2005-01-13 The University Of Manchester Microfluidic method and device
US6844377B1 (en) 1998-09-30 2005-01-18 Basf Aktiengesellschaft Polymer particles containing dye
US20050019776A1 (en) 2002-06-28 2005-01-27 Callow Matthew James Universal selective genome amplification and universal genotyping system
WO2005010145A2 (en) 2003-07-05 2005-02-03 The Johns Hopkins University Method and compositions for detection and enumeration of genetic variations
US20050032238A1 (en) 2003-08-07 2005-02-10 Nanostream, Inc. Vented microfluidic separation devices and methods
WO2005011867A2 (en) 2003-07-31 2005-02-10 Handylab, Inc. Processing particle-containing samples
JP2005037346A (en) 2003-06-25 2005-02-10 Aisin Seiki Co Ltd Micro fluid control system
US20050037397A1 (en) 2001-03-28 2005-02-17 Nanosphere, Inc. Bio-barcode based detection of target analytes
US20050042639A1 (en) 2002-12-20 2005-02-24 Caliper Life Sciences, Inc. Single molecule amplification and detection of DNA length
US20050048467A1 (en) 2001-07-20 2005-03-03 Sastry Jagannadha K. Method and compositions relating to hpv-associated pre-cancerous and cancerous growths, including cin
US20050048669A1 (en) 2003-08-26 2005-03-03 Nanostream, Inc. Gasketless microfluidic device interface
WO2005021151A1 (en) 2003-08-27 2005-03-10 President And Fellows Of Harvard College Electronic control of fluidic species
WO2005023427A1 (en) 2003-09-05 2005-03-17 Stokes Bio Limited A microfluidic analysis system
US6872250B2 (en) 2001-06-08 2005-03-29 Syrrx, Inc. Microvolume crystallization method employing multiple lumens
WO2005028674A2 (en) 2003-09-22 2005-03-31 Trisogen Biotechnology Limited Partnership Methods and kits useful for detecting an alteration in a locus copy number
US20050079501A1 (en) 2002-01-25 2005-04-14 Hisashi Koike Method and apparatus for detecting nucleic acid data
US20050084923A1 (en) 2001-09-14 2005-04-21 Peter-Juergen Mueller Methods for cultivating and analyzing microbial individual cell cultures
US20050087122A1 (en) 2002-05-09 2005-04-28 Ismagliov Rustem F. Device and method for pressure-driven plug transport and reaction
US20050095611A1 (en) 2003-05-02 2005-05-05 Chan Daniel W. Identification of biomarkers for detecting pancreatic cancer
US6890487B1 (en) 1999-09-30 2005-05-10 Science & Technology Corporation ©UNM Flow cytometry for high throughput screening
US20050100895A1 (en) 2000-09-18 2005-05-12 Waldman Scott A. Compositions and methods for identifying and targeting stomach and esophageal cancer cells
WO2005041884A2 (en) 2003-10-31 2005-05-12 Engineered Release Systems, Inc Polymer-based microstructures
US20050103690A1 (en) 2003-11-19 2005-05-19 Aisin Seiki Kabushiki Kaisha Micro liquid control system
US6897018B1 (en) 1998-02-25 2005-05-24 The United States Of America As Represented By The Department Of Health And Human Services DLC-1 gene deleted in cancers
WO2005049787A2 (en) 2003-11-24 2005-06-02 Yeda Research And Development Co.Ltd. Compositions and methods for in vitro sorting of molecular and cellular libraries
US20050123937A1 (en) 2003-03-07 2005-06-09 Thorp H. H. Methods for the electrochemical detection of target compounds
US6905844B2 (en) 2000-11-28 2005-06-14 Kim Jin-Woo Human cervical cancer 2 protooncogene and protein encoded therein
US20050129582A1 (en) 2003-06-06 2005-06-16 Micronics, Inc. System and method for heating, cooling and heat cycling on microfluidic device
US20050152908A1 (en) 2003-11-03 2005-07-14 Genenews Inc. Liver cancer biomarkers
JP2005192944A (en) 2004-01-09 2005-07-21 Keio Gijuku Telemedicine system for pigmentation site
US20050161669A1 (en) 2002-08-02 2005-07-28 Jovanovich Stevan B. Integrated system with modular microfluidic components
US20050170373A1 (en) 2003-09-10 2005-08-04 Althea Technologies, Inc. Expression profiling using microarrays
US20050169797A1 (en) 2002-05-08 2005-08-04 Mitsuaki Oshima Biomolecular substrate, method of testing or diagnosis with use thereof and apparatus therefor
US20050170431A1 (en) 2003-02-28 2005-08-04 Plexxikon, Inc. PYK2 crystal structure and uses
US6926313B1 (en) 2003-04-02 2005-08-09 Sandia National Laboratories High pressure capillary connector
US20050183995A1 (en) 2002-04-17 2005-08-25 Cytonome, Inc. Method and apparatus for sorting particles
US6936417B2 (en) 1999-02-22 2005-08-30 Aros Applied Biotechnology Aps Gene expression in bladder tumors
US6942978B1 (en) 1999-03-03 2005-09-13 The Board Of Trustees Of The University Of Arkansas Transmembrane serine protease overexpressed in ovarian carcinoma and uses thereof
EP1574586A2 (en) * 2004-03-12 2005-09-14 Samsung Electronics Co., Ltd. Method and apparatus for amplifying nucleic acids
US20050208495A1 (en) 2002-09-17 2005-09-22 Joseph Richard A Real-time detection of nucleic acid reactions
US20050208529A1 (en) 2003-12-01 2005-09-22 Lars Winther Methods and compositions for immuno-histochemical detection
US20050207940A1 (en) 2003-08-28 2005-09-22 Butler William F Methods and apparatus for sorting cells using an optical switch in a microfluidic channel network
US6949342B2 (en) 2001-12-21 2005-09-27 Whitehead Institute For Biomedical Research Prostate cancer diagnosis and outcome prediction by expression analysis
US20050214173A1 (en) 2004-01-25 2005-09-29 Fluidigm Corporation Integrated chip carriers with thermocycler interfaces and methods of using the same
US20050221341A1 (en) 2003-10-22 2005-10-06 Shimkets Richard A Sequence-based karyotyping
US20050221339A1 (en) 2004-03-31 2005-10-06 Medical Research Council Harvard University Compartmentalised screening by microfluidic control
US20050227264A1 (en) * 2004-01-28 2005-10-13 Nobile John R Nucleic acid amplification with continuous flow emulsion
WO2005103106A1 (en) 2004-04-23 2005-11-03 Eugenia Kumacheva Method of producing polymeric particles with selected size, shape, morphology and composition
US20050251049A1 (en) 1996-11-19 2005-11-10 Astron Clinica Limited Method of and apparatus for investigating tissue histology
US20050248066A1 (en) 2000-05-22 2005-11-10 Esteban Miguel A S Outer surfaces of sun visors
US6964847B1 (en) 1999-07-14 2005-11-15 Packard Biosciences Company Derivative nucleic acids and uses thereof
US20050260566A1 (en) 2004-03-24 2005-11-24 Tripath Imaging, Inc. Methods and compositions for the detection of cervical disease
US6974667B2 (en) 2000-06-14 2005-12-13 Gene Logic, Inc. Gene expression profiles in liver cancer
WO2005118138A1 (en) 2004-06-04 2005-12-15 Crystal Vision Microsystems Llc Device and process for continuous on-chip flow injection analysis
WO2005118867A2 (en) 2004-06-01 2005-12-15 The Regents Of The University Of California Microfabricated integrated dna analysis system
US20060003439A1 (en) 2004-07-02 2006-01-05 Ismagilov Rustem F Microfluidic system
US20060003429A1 (en) 1999-01-29 2006-01-05 Frost John W Biocatalytic synthesis of quinic acid and conversion to hydroquinone
WO2006002641A1 (en) 2004-07-02 2006-01-12 Versamatrix A/S Spherical radiofrequency-encoded beads
US20060008824A1 (en) 2004-05-20 2006-01-12 Leland Stanford Junior University Methods and compositions for clonal amplification of nucleic acid
WO2006009657A1 (en) 2004-06-17 2006-01-26 Essilor International (Compagnie Generale D'optique) Progressive addition lenses with reduced unwanted astigmatism
US6998232B1 (en) 1999-09-27 2006-02-14 Quark Biotech, Inc. Methods of diagnosing bladder cancer
US20060035386A1 (en) 2002-12-02 2006-02-16 Nec Corporation Fine particle handling unit, chip and sensor mounted with same, and methods for separating, capturing and sensing protein
US20060040297A1 (en) * 2003-01-29 2006-02-23 Leamon John H Methods of amplifying and sequencing nucleic acids
US20060040197A1 (en) 2004-08-20 2006-02-23 Kabushiki Kaisha Toshiba Toner and fixing method
US20060046257A1 (en) 2004-01-27 2006-03-02 Sarah Pollock Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis of lung cancer
US20060051329A1 (en) 2004-08-27 2006-03-09 The Regents Of The University Of California Microfluidic device for the encapsulation of cells with low and high cell densities
WO2006027757A2 (en) 2004-09-09 2006-03-16 Institut Curie Microfluidic device using a collinear electric field
US20060068398A1 (en) 2004-09-24 2006-03-30 Cepheid Universal and target specific reagent beads for nucleic acid amplification
US7022472B2 (en) 1998-10-22 2006-04-04 Diadexus, Inc. Mutations in human MLH1 and human MSH2 genes useful in diagnosing colorectal cancer
US20060078893A1 (en) 2004-10-12 2006-04-13 Medical Research Council Compartmentalised combinatorial chemistry by microfluidic control
US20060078888A1 (en) 2004-10-08 2006-04-13 Medical Research Council Harvard University In vitro evolution in microfluidic systems
US20060078475A1 (en) 2004-07-29 2006-04-13 Yu-Chong Tai Modular microfluidic packaging system
WO2006042303A2 (en) 2004-10-12 2006-04-20 Ambion, Inc. Methods and compositions for analyzing ribonucleic acids
US20060094119A1 (en) 2004-10-29 2006-05-04 Ismagilov Rustem F Microfluidic system
US20060096923A1 (en) 2002-05-24 2006-05-11 Fraunhoffer-Gesellschaft Zur Forderung Der Angewan Method for transferring heterogeneous liquids in microchannels without the occurrence of mixing
US20060100788A1 (en) 2004-07-14 2006-05-11 Invitrogen Corporation Collections of matched biological reagents and methods for identifying matched reagents
US20060105170A1 (en) 2002-08-29 2006-05-18 Isis Innovation Limited Magnetic particle and process for preparation
US7049072B2 (en) 2000-06-05 2006-05-23 University Of South Florida Gene expression analysis of pluri-differentiated mesenchymal progenitor cells and methods for diagnosing a leukemic disease state
US20060110759A1 (en) 2004-11-05 2006-05-25 Regents Of The University Of California Biomarkers for prostate cancer metastasis
US20060108012A1 (en) 2002-11-14 2006-05-25 Barrow David A Microfluidic device and methods for construction and application
US20060115821A1 (en) 2003-06-26 2006-06-01 Richard Einstein Prostate specific genes and the use thereof as targets for prostate cancer therapy
US7056674B2 (en) 2003-06-24 2006-06-06 Genomic Health, Inc. Prediction of likelihood of cancer recurrence
US7057026B2 (en) 2001-12-04 2006-06-06 Solexa Limited Labelled nucleotides
US7068874B2 (en) 2000-11-28 2006-06-27 The Regents Of The University Of California Microfluidic sorting device
US20060147909A1 (en) 2001-05-31 2006-07-06 Markus Rarbach Microstructures and use thereof for the directed evolution of biomolecules
US7078180B2 (en) 2001-09-05 2006-07-18 The Children's Hospital Of Philadelphia Methods and compositions useful for diagnosis, staging, and treatment of cancers and tumors
US20060160762A1 (en) 2004-12-13 2006-07-20 Children's Medical Center Corporation Methods for the treatment, diagnosis, and prognosis of cancer
US7081192B1 (en) 2000-08-08 2006-07-25 Aviva Biosciences Corporation Methods for manipulating moieties in microfluidic systems
US7081340B2 (en) 2002-03-13 2006-07-25 Genomic Health, Inc. Gene expression profiling in biopsied tumor tissues
WO2006078841A1 (en) 2005-01-21 2006-07-27 President And Fellows Of Harvard College Systems and methods for forming fluidic droplets encapsulated in particles such as colloidal particles
WO2006076810A1 (en) 2005-01-21 2006-07-27 Perceptronix Medical Inc. Method And Apparatus For Measuring Cancerous Changes From Reflectance Spectral Measurements Obtained During Endoscopic Imaging
US20060169800A1 (en) 1999-06-11 2006-08-03 Aradigm Corporation Aerosol created by directed flow of fluids and devices and methods for producing same
US20060177832A1 (en) 2005-02-10 2006-08-10 Sydney Brenner Genetic analysis by sequence-specific sorting
US7090983B1 (en) 1999-09-10 2006-08-15 Takashi Muramatsu Methods for detecting early cancer
US20060195269A1 (en) 2004-02-25 2006-08-31 Yeatman Timothy J Methods and systems for predicting cancer outcome
WO2006096571A2 (en) 2005-03-04 2006-09-14 President And Fellows Of Harvard College Method and apparatus for forming multiple emulsions
US7115230B2 (en) 2003-06-26 2006-10-03 Intel Corporation Hydrodynamic focusing devices
US20060223127A1 (en) 2002-12-18 2006-10-05 Ciphergen Biosystems, Inc. Serum biomarkers in lung cancer
US7118910B2 (en) 2001-11-30 2006-10-10 Fluidigm Corporation Microfluidic device and methods of using same
US20060234264A1 (en) 2005-03-14 2006-10-19 Affymetrix, Inc. Multiplex polynucleotide synthesis
US20060234259A1 (en) 2005-02-25 2006-10-19 Rubin Mark A Biomarkers for predicting prostate cancer progression
US20060234254A1 (en) 2004-11-08 2006-10-19 Sungwhan An Colon cancer biomarker discovery
US20060245971A1 (en) 2005-05-02 2006-11-02 Burns Andrew A Photoluminescent silica-based sensors and methods of use
US20060246431A1 (en) 2001-12-07 2006-11-02 Wamadiva Balachandran Test apparatus
US20060247532A1 (en) 2005-05-02 2006-11-02 Nirmala Ramanujam Method for extraction of optical properties from diffuse reflectance spectra
US20060252057A1 (en) 2004-11-30 2006-11-09 Mitch Raponi Lung cancer prognostics
US20060257893A1 (en) * 2005-02-18 2006-11-16 Toru Takahashi Devices and methods for monitoring genomic DNA of organisms
US20060258841A1 (en) 2003-01-17 2006-11-16 Josef Michl Pancreatic cancer associated antigen, antibody thereto, and diagnostic and treatment methods
US20060263888A1 (en) 2000-06-02 2006-11-23 Honeywell International Inc. Differential white blood count on a disposable card
US20060269558A1 (en) 1998-04-27 2006-11-30 Murphy Gerald P Nr-CAM gene, nucleic acids and nucleic acid products for therapeutic and diagnostic uses for tumors
US20060269971A1 (en) 2003-09-26 2006-11-30 Mount Sinai Hospital Methods for detecting prostate cancer
US20060269934A1 (en) 2005-03-16 2006-11-30 Applera Corporation Compositions and methods for clonal amplification and analysis of polynucleotides
US20060281089A1 (en) 2003-06-12 2006-12-14 University Of Manitoba Methods for detecting cancer and monitoring cancer progression
US20060281098A1 (en) 2005-06-14 2006-12-14 Xin Miao Method and kits for multiplex hybridization assays
US20060286570A1 (en) 2003-09-09 2006-12-21 Rowlen Kathy L Use of photopolymerization for amplification and detection of a molecular recognition event
US7153700B1 (en) 1999-03-26 2006-12-26 Dana-Farber Cancer Institute, Inc. Methods and compositions for diagnosing and predicting the behavior of cancer
US7156917B2 (en) 2003-05-22 2007-01-02 Hideaki Moriyama Apparatus and method for growing crystal, and apparatus and method for analyzing crystal
EP1741482A2 (en) 2001-02-23 2007-01-10 Japan Science and Technology Agency Process and apparatus for producing microcapsules
US20070009954A1 (en) 2001-11-28 2007-01-11 Bio-Rad Laboratories, Inc. Parallel polymorphism scoring by amplification and error correction
US20070009914A1 (en) 2005-07-07 2007-01-11 Wallace Robert B Labeled complimentary oligonucleotides to detect oligonucleotide-linked ligands
US7163801B2 (en) 1999-09-01 2007-01-16 The Burnham Institute Methods for determining the prognosis for cancer patients using tucan
US20070016078A1 (en) 2004-12-06 2007-01-18 Hoyt Clifford C Systems and methods for in-vivo optical imaging and measurement
US20070020617A1 (en) 1998-08-07 2007-01-25 Cellay, Llc C/O One Cell Systems, Inc. Gel microdrops in genetic analysis
US7171311B2 (en) 2001-06-18 2007-01-30 Rosetta Inpharmatics Llc Methods of assigning treatment to breast cancer patients
US7169560B2 (en) 2003-11-12 2007-01-30 Helicos Biosciences Corporation Short cycle methods for sequencing polynucleotides
US20070026439A1 (en) 2005-07-15 2007-02-01 Applera Corporation Fluid processing device and method
WO2007012638A1 (en) 2005-07-25 2007-02-01 Commissariat A L'energie Atomique Method for controlling communication between two electrowetting zones, device comprising zones capable of being isolated from one another and method for making such a device
US20070031829A1 (en) 2002-09-30 2007-02-08 Hideyuki Yasuno Oligonucleotides for genotyping thymidylate synthase gene
US20070042400A1 (en) 2003-11-10 2007-02-22 Choi K Y Methods of preparing nucleic acid for detection
WO2007021343A2 (en) 2005-05-18 2007-02-22 Cornell Research Foundation, Inc. Pharmacokinetic-based culture system with biological barriers
US20070042419A1 (en) 1996-05-29 2007-02-22 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
US20070039866A1 (en) 2005-08-22 2007-02-22 Schroeder Benjamin G Device, system, and method for depositing processed immiscible-fluid-discrete-volumes
US20070048744A1 (en) 2005-08-26 2007-03-01 Stanley Lapidus Single molecule sequencing of captured nucleic acids
US20070053896A1 (en) 2003-09-05 2007-03-08 Royal Women's Hospital Diagnostic marker for ovarian cancer
US20070054119A1 (en) 2005-03-04 2007-03-08 Piotr Garstecki Systems and methods of forming particles
WO2007026884A1 (en) 2005-09-02 2007-03-08 Pola Chemical Industries Inc. Method of evaluating skin conditions and method of estimating skin thickness
US20070056853A1 (en) 2005-09-15 2007-03-15 Lucnet Technologies Inc. Micro-chemical mixing
WO2007030501A2 (en) 2005-09-08 2007-03-15 Brandeis University Microfluidic manipulation of fluids and reactions
US7198899B2 (en) 2002-06-03 2007-04-03 Chiron Corporation Use of NRG4, or inhibitors thereof, in the treatment of colon and pancreatic cancers
US7204431B2 (en) 2003-10-31 2007-04-17 Agilent Technologies, Inc. Electrospray ion source for mass spectroscopy
US20070111303A1 (en) 2005-09-01 2007-05-17 Hiroshi Inoue Method and molecular diagnostic device for detection, analysis and identification of genomic DNA
US20070123430A1 (en) 2005-08-18 2007-05-31 David Pasquier Well fluid comprising a fluorinated liquid phase
US20070120899A1 (en) 2004-09-30 2007-05-31 Ngk Insulators, Ltd. Liquid drop discharge piezoelectric device
US7229770B1 (en) 1998-10-01 2007-06-12 The Regents Of The University Of California YKL-40 as a marker and prognostic indicator for cancers
US7229760B2 (en) 2000-03-24 2007-06-12 Micromet Ag mRNA amplification
US20070142720A1 (en) 2004-05-24 2007-06-21 Trent Ridder Apparatus and methods for mitigating the effects of foreign interferents on analyte measurements in spectroscopy
US20070156037A1 (en) 2004-06-17 2007-07-05 The Regents Of The University Of Californa Time-resolved non-invasive optometric device for medical diagnostic
US20070154889A1 (en) 2004-06-25 2007-07-05 Veridex, Llc Methods and reagents for the detection of melanoma
WO2007081385A2 (en) 2006-01-11 2007-07-19 Raindance Technologies, Inc. Microfluidic devices and methods of use in the formation and control of nanoreactors
US20070172873A1 (en) 2006-01-23 2007-07-26 Sydney Brenner Molecular counting
JP2007190364A (en) 2005-09-23 2007-08-02 Astron Clinica Ltd Image processing method and apparatus
US20070184439A1 (en) 2003-07-17 2007-08-09 Guilford Parry J Markers for detection of gastric cancer
WO2007089541A2 (en) 2006-01-27 2007-08-09 President And Fellows Of Harvard College Fluidic droplet coalescence
WO2007092473A2 (en) 2006-02-02 2007-08-16 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive fetal genetic screening by digital analysis
WO2007114794A1 (en) 2006-03-31 2007-10-11 Nam Trung Nguyen Active control for droplet-based microfluidics
US7282337B1 (en) 2006-04-14 2007-10-16 Helicos Biosciences Corporation Methods for increasing accuracy of nucleic acid sequencing
US20070243634A1 (en) 2006-04-18 2007-10-18 Pamula Vamsee K Droplet-based surface modification and washing
US20070241068A1 (en) 2006-04-13 2007-10-18 Pamula Vamsee K Droplet-based washing
US20070242105A1 (en) 2006-04-18 2007-10-18 Vijay Srinivasan Filler fluids for droplet operations
WO2007123744A2 (en) 2006-03-31 2007-11-01 Solexa, Inc. Systems and devices for sequence by synthesis analysis
US7291462B2 (en) 1998-02-20 2007-11-06 The University Of Arkansas For Medical Sciences TADG-15: an extracellular serine protease overexpressed in carcinomas
US20070259368A1 (en) 2006-05-03 2007-11-08 Genomictree, Inc. Gastric cancer biomarker discovery
US20070259351A1 (en) 2006-05-03 2007-11-08 James Chinitz Evaluating Genetic Disorders
US20070269804A1 (en) 2004-06-19 2007-11-22 Chondrogene, Inc. Computer system and methods for constructing biological classifiers and uses thereof
WO2007133710A2 (en) 2006-05-11 2007-11-22 Raindance Technologies, Inc. Microfluidic devices and methods of use thereof
US7300765B2 (en) 2002-04-02 2007-11-27 Ucb Pharma S.A. SC6 for diagnosis of cancers
US20070275415A1 (en) 2006-04-18 2007-11-29 Vijay Srinivasan Droplet-based affinity assays
WO2007138178A2 (en) 2006-05-30 2007-12-06 Centre National De La Recherche Scientifique Method for treating drops in a microfluid circuit
WO2007140015A2 (en) 2006-05-26 2007-12-06 Althea Technologies, Inc Biochemical analysis of partitioned cells
US7308364B2 (en) 2001-11-07 2007-12-11 The University Of Arkansas For Medical Sciences Diagnosis of multiple myeloma on gene expression profiling
US20070292869A1 (en) 2006-03-02 2007-12-20 Ppd Biomarker Discovery Sciences, Llc Compositions and Methods for Analyzing Renal Cancer
US7314721B2 (en) 2000-01-21 2008-01-01 Ludwig Institute For Cancer Research Small cell lung cancer associated antigens and uses therefor
US20080003571A1 (en) 2005-02-01 2008-01-03 Mckernan Kevin Reagents, methods, and libraries for bead-based sequencing
US20080004436A1 (en) 2004-11-15 2008-01-03 Yeda Research And Development Co. Ltd. At The Weizmann Institute Of Science Directed Evolution and Selection Using in Vitro Compartmentalization
US7316906B2 (en) 1999-10-08 2008-01-08 The Feinstein Institute For Medical Research CD38 as a prognostic indicator in B cell chronic lymphocytic leukemia
US20080009005A1 (en) 2006-02-09 2008-01-10 Kruk Patricia A Detection of cancer by elevated levels of BCL-2
US20080014590A1 (en) 2004-01-27 2008-01-17 Compugen Ltd. Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis of prostate cancer
US20080020940A1 (en) 2006-07-24 2008-01-24 Miraculins Inc. Biomarkers for use in the diagnosis and treatment of colorectal cancer
US20080021330A1 (en) 2006-07-21 2008-01-24 Samsung Electronics Co., Ltd. Apparatus, method and optical sensor module using a tilter for body fat measurement
US7323309B2 (en) 2000-03-28 2008-01-29 Northwestern University Bio-barcodes based on oligonucleotide-modified particles
US7326529B2 (en) 1999-12-06 2008-02-05 Diadexus, Inc. Method of diagnosing, monitoring, staging, imaging and treating prostate cancer
US20080032413A1 (en) 2004-04-12 2008-02-07 Byeang-Hyean Kim Oligonucleotide For Detecting Target Dna Or Rna
US20080038754A1 (en) 2004-07-14 2008-02-14 The Regents Of The University Of California Biomarkers for Early Detection of Ovarian Cancer
US7332280B2 (en) 2003-10-14 2008-02-19 Ronald Levy Classification of patients having diffuse large B-cell lymphoma based upon gene expression
US7332590B2 (en) 2001-08-16 2008-02-19 The United States Of America As Represented By The Department Of Health And Human Services Molecular characteristics of non-small cell lung cancer
WO2008021123A1 (en) 2006-08-07 2008-02-21 President And Fellows Of Harvard College Fluorocarbon emulsion stabilizing surfactants
US20080044828A1 (en) 2004-02-04 2008-02-21 Kwok Tim T CUDR as biomarker for cancer progression and therapeutic response
US20080050378A1 (en) 2004-03-23 2008-02-28 Oncotherapy Science, Inc. Method for Diagnosing Non-Small Cell Lung Cancer
US20080050723A1 (en) 2006-08-23 2008-02-28 Nabil Belacel Molecular method for diagnosis of colon cancer
US20080058432A1 (en) 2006-03-03 2008-03-06 Yixin Wang Molecular assay to predict recurrence of Duke's B colon cancer
US20080057514A1 (en) 2006-09-06 2008-03-06 Vanderbilt University Methods of screening for gastrointestinal cancer
US20080053205A1 (en) 2006-04-18 2008-03-06 Pollack Michael G Droplet-based particle sorting
US7341211B2 (en) 2002-02-04 2008-03-11 Universidad De Sevilla Device for the production of capillary jets and micro-and nanometric particles
US20080063227A1 (en) 2006-09-07 2008-03-13 Kristin Rohrseitz Method for adapting a hearing aid using a genetic feature
US20080064047A1 (en) 2005-01-28 2008-03-13 Zetter Bruce R Methods for diagnosis and prognosis of epithelial cancers
US7348142B2 (en) 2002-03-29 2008-03-25 Veridex, Lcc Cancer diagnostic panel
US20080081333A1 (en) 2006-05-26 2008-04-03 University Of Maryland, Baltimore Methylated promoters as biomarkers of colon cancer
US20080081330A1 (en) 2006-09-28 2008-04-03 Helicos Biosciences Corporation Method and devices for analyzing small RNA molecules
US7358231B1 (en) 2005-12-01 2008-04-15 Applera Corporation Pancreatic cancer secreted targets and uses thereof
US7361474B2 (en) 2003-02-24 2008-04-22 United States Of America As Represented By The Department Of Veterans Affairs Serum macrophage migration inhibitory factor (MIF) as marker for prostate cancer
US20080092973A1 (en) 2006-10-20 2008-04-24 Tai-Sol Electronics Co., Ltd. Flexible heat pipe
US7364862B2 (en) 1998-10-19 2008-04-29 Diadexus, Inc. Method of diagnosing, monitoring, staging, imaging and treating prostate cancer
WO2008052138A2 (en) 2006-10-25 2008-05-02 The Regents Of The University Of California Inline-injection microdevice and microfabricated integrated dna analysis system using same
US7368255B2 (en) 2001-01-29 2008-05-06 Suk-Chul Bae RUNX3 gene showing anti-tumor activity and use thereof
US20080113340A1 (en) 2003-07-18 2008-05-15 Georgetown University Diagnosis and treatment of cervical cancer
US20080118462A1 (en) 2005-01-07 2008-05-22 The John Hopkins University Biomarkers for Melanoma
US7378280B2 (en) 2000-11-16 2008-05-27 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
US7378233B2 (en) 2003-04-12 2008-05-27 The Johns Hopkins University BRAF mutation T1796A in thyroid cancers
US20080138806A1 (en) 2006-12-08 2008-06-12 National Taiwan University Biomarkers and detection methods for gastric diseases
US7390463B2 (en) 2001-09-07 2008-06-24 Corning Incorporated Microcolumn-based, high-throughput microfluidic device
US7393665B2 (en) 2005-02-10 2008-07-01 Population Genetics Technologies Ltd Methods and compositions for tagging and identifying polynucleotides
US7393634B1 (en) 1999-10-12 2008-07-01 United States Of America As Represented By The Secretary Of The Air Force Screening for disease susceptibility by genotyping the CCR5 and CCR2 genes
JP2008153669A (en) 2006-12-15 2008-07-03 Cree Inc Reflective mounting substrate for light emitting diodes
US20080166793A1 (en) 2007-01-04 2008-07-10 The Regents Of The University Of California Sorting, amplification, detection, and identification of nucleic acid subsequences in a complex mixture
US20080171078A1 (en) 2007-01-12 2008-07-17 Mark Gray Uniformly sized liposomes
US20080176236A1 (en) 2006-11-15 2008-07-24 Ming Sound Tsao Materials and methods for prognosing lung cancer survival
US7405002B2 (en) 2004-08-04 2008-07-29 Agency For Science, Technology And Research Coated water-soluble nanoparticles comprising semiconductor core and silica coating
US20080181850A1 (en) 2007-01-29 2008-07-31 Northwestern University Biomarkers For Prostate Cancer
WO2008097559A2 (en) 2007-02-06 2008-08-14 Brandeis University Manipulation of fluids and reactions in microfluidic systems
US7416851B2 (en) 2004-11-08 2008-08-26 Institut Pasteur Method of diagnosis/prognosis of human chronic lymphocytic leukemia comprising the profiling of LPL/ADAM genes
US20080206756A1 (en) 2003-07-18 2008-08-28 California Pacific Medical Center Biomarker panel for colorectal cancer
US20080213377A1 (en) 2006-12-08 2008-09-04 Bhatia Sangeeta N Delivery of Nanoparticles and/or Agents to Cells
US20080222741A1 (en) 2001-08-02 2008-09-11 The Regents Of The University Of Michigan Expression Profile Of Prostate Cancer
US20080216563A1 (en) 2006-09-29 2008-09-11 Reed Wayne F Methods and devices for simultaneously monitoring the characteristics of microscopic particles in suspension and the characteristics of soluble components during reactions
US20080220986A1 (en) 2006-08-24 2008-09-11 Niall Anthony Gormley Method for retaining even coverage of short insert libraries
WO2008115626A2 (en) 2007-02-05 2008-09-25 Microchip Biotechnologies, Inc. Microfluidic and nanofluidic devices, systems, and applications
US20080234138A1 (en) 2006-12-08 2008-09-25 Shaughnessy John D TP53 gene expression and uses thereof
US7432064B2 (en) 1998-10-19 2008-10-07 Diadexus, Inc. Method of diagnosing, monitoring, staging, imaging and treating prostate cancer
WO2008121342A2 (en) 2007-03-28 2008-10-09 President And Fellows Of Harvard College Emulsions and techniques for formation
US20080261295A1 (en) 2007-04-20 2008-10-23 William Frank Butler Cell Sorting System and Methods
US7442507B2 (en) 2005-01-24 2008-10-28 New York University School Of Medicine Methods for detecting circulating mutant BRAF DNA
US20080268473A1 (en) 2005-02-17 2008-10-30 Moses Marsha A Adamts-7 as a Biomarker for Cancers of Epithelial Origin
US20080269157A1 (en) 2006-10-10 2008-10-30 The Henry F. Jackson Foundation For Military Medicine Prostate cancer-specific alterations in ERG gene expression and detection and treatment methods based on those alterations
WO2008130623A1 (en) 2007-04-19 2008-10-30 Brandeis University Manipulation of fluids, fluid components and reactions in microfluidic systems
US20080274513A1 (en) 2005-05-11 2008-11-06 Shenderov Alexander D Method and Device for Conducting Biochemical or Chemical Reactions at Multiple Temperatures
US20080274908A1 (en) 2007-05-04 2008-11-06 Dermtech International Diagnosis of melanoma by nucleic acid analysis
WO2008134153A1 (en) 2007-04-23 2008-11-06 Advanced Liquid Logic, Inc. Bead-based multiplexed analytical methods and instrumentation
US7449303B2 (en) 2003-05-02 2008-11-11 Health Research, Inc. Use of JAG2 expression in diagnosis of plasma cell disorders
US20080280302A1 (en) 2007-05-09 2008-11-13 The Regents Of The University Of California Multigene diagnostic assay for malignant thyroid neoplasm
US20080280285A1 (en) 2005-05-11 2008-11-13 Chen Zongyuan G Systems and Methods For Testing using Microfluidic Chips
US20080286801A1 (en) 2005-12-21 2008-11-20 Carlos Buesa Arjol Method for the analysis of differential expression in colorectal cancer
US20080286811A1 (en) 2005-02-18 2008-11-20 Moses Marsha A Cyr61 as a Biomarker for Diagnosis and Prognosis of Cancers of Epithelial Origin
US20080286199A1 (en) 2005-02-16 2008-11-20 Livingston David M Methods of Detecting Ovarian Cancer
US20080293578A1 (en) 2001-11-07 2008-11-27 Shaugnessy John D Diagnosis, prognosis and identification of potential therapeutic targets of multiple myeloma based on gene expression profiling
US20080299565A1 (en) 2005-12-12 2008-12-04 Schneider Thomas D Probe for Nucleic Acid Sequencing and Methods of Use
US20080305482A1 (en) 2006-12-21 2008-12-11 Gen-Probe Incorporated Methods and compositions for nucleic acid amplification
US20080311570A1 (en) 2007-06-15 2008-12-18 National Defense Medical Center Cancer screening method
US20080311604A1 (en) 2005-11-02 2008-12-18 Elting James J Methods for Prediction and Prognosis of Cancer, and Monitoring Cancer Therapy
US7468271B2 (en) 2005-04-06 2008-12-23 President And Fellows Of Harvard College Molecular characterization with carbon nanotube control
US20090004687A1 (en) 2007-06-29 2009-01-01 Mansfield Brian C Predictive markers for ovarian cancer
US7473530B2 (en) 2005-05-04 2009-01-06 Wayne State University Method to detect lung cancer
US7473531B1 (en) 2003-08-08 2009-01-06 Colora Corporation Pancreatic cancer targets and uses thereof
US20090009855A1 (en) 2005-03-04 2009-01-08 Japan Science And Technology Agency Wide-band optical amplifier
US20090017463A1 (en) 2007-07-10 2009-01-15 Vanderbilt University Methods for predicting prostate cancer recurrence
US7479371B2 (en) 2002-04-09 2009-01-20 Tokai University Method of judging leukemia, pre-leukemia or aleukemic malignant blood disease and diagnostic therefor
US7479370B2 (en) 2003-09-08 2009-01-20 Health Research, Inc. Detection of 13q14 chromosomal alterations
EP2017910A1 (en) 2007-07-20 2009-01-21 BIOTRONIK CRM Patent AG Active element and battery and method for production of same
US20090023137A1 (en) 2004-07-16 2009-01-22 Oncomethylome Sciences S.A. ESR1 and Cervical Cancer
US20090021728A1 (en) 2007-06-21 2009-01-22 Gen-Probe Incorporated Multi-Channel Optical Measurement Instrument
US7482129B2 (en) 2004-05-04 2009-01-27 Institute Of Virology, Slovak Academy Of Sciences MN/CA IX/CA9 and Renal Cancer Prognosis
WO2009015296A1 (en) 2007-07-24 2009-01-29 The Regents Of The University Of California Microfabricated dropley generator
US20090029372A1 (en) 2007-05-14 2009-01-29 Kobenhavns Universitet Adam12 as a biomarker for bladder cancer
US20090026082A1 (en) 2006-12-14 2009-01-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20090042737A1 (en) 2007-08-09 2009-02-12 Katz Andrew S Methods and Devices for Correlated, Multi-Parameter Single Cell Measurements and Recovery of Remnant Biological Material
US20090053732A1 (en) 2007-07-16 2009-02-26 Ophir Vermesh Microfluidic devices, methods and systems for detecting target molecules
US20090060797A1 (en) 2002-12-30 2009-03-05 The Regents Of The University Of California Fluid control structures in microfluidic devices
WO2009029229A2 (en) 2007-08-24 2009-03-05 President And Fellows Of Harvard College Ferrofluid emulsions, particles, and systems and methods for making and using the same
US20090062144A1 (en) 2007-04-03 2009-03-05 Nancy Lan Guo Gene signature for prognosis and diagnosis of lung cancer
US7501244B2 (en) 2001-02-21 2009-03-10 Novartis Vaccines And Diagnostics, Inc. Determining prognosis of colon or breast cancer by measuring TTK expression
US20090069194A1 (en) 2007-09-07 2009-03-12 Fluidigm Corporation Copy number variation determination, methods and systems
US20090068170A1 (en) 2007-07-13 2009-03-12 President And Fellows Of Harvard College Droplet-based selection
US7504214B2 (en) 2003-09-19 2009-03-17 Biotheranostics, Inc. Predicting outcome with tamoxifen in breast cancer
US20090075307A1 (en) 2006-01-27 2009-03-19 Tripath Imaging, Inc. Methods for identifying patients with an increased likelihood of having ovarian cancer and compositions therefor
US20090075265A1 (en) 2007-02-02 2009-03-19 Orion Genomics Llc Gene methylation in thyroid cancer diagnosis
US20090075311A1 (en) 2005-12-21 2009-03-19 Johann Karl Assessing colorectal cancer by measuring hemoglobin and m2-pk in a stool sample
US7507532B2 (en) 2004-03-08 2009-03-24 Medigen Biotechnology Corporation Cancer specific gene MH15
US7507541B2 (en) 1999-10-28 2009-03-24 Agensys, Inc. 36P6D5: secreted tumor antigen
WO2009037266A2 (en) 2007-09-17 2009-03-26 Universite Louis Pasteur Method for detecting or quantifying a truncating mutation
US20090081685A1 (en) 2004-07-09 2009-03-26 Tripath Imaging, Inc. Methods and compositions for the detection of ovarian disease
US20090081237A1 (en) 2007-03-12 2009-03-26 Dana-Farber Cancer Institute Prognostic, diagnostic, and cancer therapeutic uses of FANCI and FANCI modulating agents
US7510842B2 (en) 2005-03-11 2009-03-31 Vermilllion, Inc. Biomarker for ovarian and endometrial cancer: hepcidin
US7510707B2 (en) 1999-12-20 2009-03-31 New York University Mt. Sinai School Of Medicine PAR, a novel marker gene for breast and prostate cancers
US20090087849A1 (en) 2007-09-06 2009-04-02 Tripath Imaging, Inc. Nucleic acid-based methods and compositions for the detection of ovarian cancer
US7514209B2 (en) 2001-06-18 2009-04-07 Rosetta Inpharmatics Llc Diagnosis and prognosis of breast cancer patients
US20090092973A1 (en) 2001-12-21 2009-04-09 Aviaradx, Inc. Grading of Breast Cancer
US20090098555A1 (en) 2007-09-26 2009-04-16 President And Fellows Of Harvard College Methods and applications for stitched dna barcodes
US20090105959A1 (en) 2007-06-01 2009-04-23 Braverman Michael S System and method for identification of individual samples from a multiplex mixture
WO2009049889A1 (en) 2007-10-16 2009-04-23 Roche Diagnostics Gmbh High resolution, high throughput hla genotyping by clonal sequencing
US7524633B2 (en) 2001-11-16 2009-04-28 The Johns Hopkins University School Of Medicine Method of detection of prostate cancer
US7527933B2 (en) 2002-11-22 2009-05-05 Ganymed Pharmaceuticals Ag Genetic products differentially expressed in tumors and the use thereof
US20090118128A1 (en) 2005-07-20 2009-05-07 Xiaohai Liu Preparation of templates for nucleic acid sequencing
US20090118158A1 (en) 2002-12-17 2009-05-07 Nastech Pharmaceutical Company Inc. Compositions for enhanced epithelial permeation of peptide yy for treating obesity
US20090124789A1 (en) 2007-11-09 2009-05-14 Yoshida Eri Polymer microparticles and production method for the same
WO2009059430A1 (en) 2007-11-07 2009-05-14 The University Of British Columbia Microfluidic device and method of using same
US20090124569A1 (en) 2007-10-12 2009-05-14 Northwestern University Inhibition and treatment of prostate cancer metastasis
US20090131353A1 (en) 2005-12-07 2009-05-21 Insel Paul A Diagnosis and Treatment of Chronic Lymphocytic Leukemia (CLL)
US20090127454A1 (en) 2006-03-24 2009-05-21 Phenomenome Discoveries Inc. Biomarkers useful for diagnosing prostate cancer, and methods thereof
US20090127589A1 (en) 2006-12-14 2009-05-21 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US7541383B2 (en) 2002-12-20 2009-06-02 Amgen Inc. Asthma and allergic inflammation modulators
US7544473B2 (en) 2006-01-23 2009-06-09 Population Genetics Technologies Ltd. Nucleic acid analysis using sequence tokens
US20090169482A1 (en) 2007-10-16 2009-07-02 Shiying Zheng Silica-cored carrier particle
WO2009085929A1 (en) 2007-12-20 2009-07-09 The Polymer Technology Group, Inc. Hybrid polyurethane block copolymers with thermoplastic processability and thermoset properties
WO2009094623A2 (en) 2008-01-24 2009-07-30 Balter, Inc. Method for discriminating between malignant and benign tissue lesions
US20090215633A1 (en) 2006-03-01 2009-08-27 Keygene N.V. High throughput sequence-based detection of snps using ligation assays
US20090226972A1 (en) 2008-01-22 2009-09-10 Neil Reginald Beer Rapid Microfluidic Thermal Cycler for Nucleic Acid Amplification
US20090226971A1 (en) 2008-01-22 2009-09-10 Neil Reginald Beer Portable Rapid Microfluidic Thermal Cycler for Extremely Fast Nucleic Acid Amplification
US20090233802A1 (en) 2007-02-02 2009-09-17 Helen Bignell Methods for indexing samples and sequencing multiple polynucleotide templates
WO2009117485A2 (en) 2008-03-18 2009-09-24 Balter, Inc. Optical method for determining morphological parameters and physiological properties of tissue
US20090246788A1 (en) 2008-04-01 2009-10-01 Roche Nimblegen, Inc. Methods and Assays for Capture of Nucleic Acids
WO2009137415A2 (en) 2008-05-03 2009-11-12 Advanced Liquid Logic, Inc. Reagent and sample preparation, loading, and storage
JP2009265751A (en) 2008-04-22 2009-11-12 Oki Electric Ind Co Ltd Character recognition device, optical character recognition system and character recognition program
US7632562B2 (en) 2005-08-04 2009-12-15 Eastman Kodak Company Universal print media
US7635562B2 (en) 2004-05-25 2009-12-22 Helicos Biosciences Corporation Methods and devices for nucleic acid sequence determination
US20090317798A1 (en) 2005-06-02 2009-12-24 Heid Christian A Analysis using microfluidic partitioning devices
US20090325217A1 (en) 2008-06-30 2009-12-31 Microbix Biosystems Inc. Method and apparatus for sorting cells
US20100003687A1 (en) 2008-07-01 2010-01-07 454 Life Sciences Corporation System and method for detection of HIV tropism variants
US20100009353A1 (en) 2006-05-18 2010-01-14 Colin Barnes Dye Compounds and the Use of their Labelled Conjugates
WO2010009365A1 (en) 2008-07-18 2010-01-21 Raindance Technologies, Inc. Droplet libraries
US20100015617A1 (en) 2006-08-31 2010-01-21 Toyo Seikan Kaisha, Ltd. Nucleic Acid Amplification Method
US20100021984A1 (en) 2008-05-23 2010-01-28 Edd Jon F Microfluidic Droplet Encapsulation
US20100035252A1 (en) 2008-08-08 2010-02-11 Ion Torrent Systems Incorporated Methods for sequencing individual nucleic acids under tension
US20100075436A1 (en) 2008-05-06 2010-03-25 Urdea Michael S Methods for use with nanoreactors
US7691576B2 (en) 2003-11-03 2010-04-06 Medical Research Council Compartmentalized self tagging
WO2010040006A1 (en) 2008-10-02 2010-04-08 Blomberg Jerome O Curbless multiple skylight system and smoke vent system
US20100086914A1 (en) 2008-10-03 2010-04-08 Roche Molecular Systems, Inc. High resolution, high throughput hla genotyping by clonal sequencing
US7698287B2 (en) 2004-09-30 2010-04-13 Microsoft Corporation Design of spreadsheet functions for working with tables of data
US20100120098A1 (en) 2008-10-24 2010-05-13 Epicentre Technologies Corporation Transposon end compositions and methods for modifying nucleic acids
WO2010056728A1 (en) 2008-11-11 2010-05-20 Helicos Biosciences Corporation Nucleic acid encoding for multiplex analysis
US20100124759A1 (en) 2008-06-27 2010-05-20 Massachusetts Institute Of Technology Microfluidic droplets for metabolic engineering and other applications
US20100129896A1 (en) 2002-12-20 2010-05-27 Caliper Life Sciences, Inc. System for differentiating the lengths of nucleic acids of interest in a sample
US20100137143A1 (en) 2008-10-22 2010-06-03 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
US20100136544A1 (en) 2007-03-07 2010-06-03 Jeremy Agresti Assays and other reactions involving droplets
US7736890B2 (en) 2003-12-31 2010-06-15 President And Fellows Of Harvard College Assay device and method
US7741130B2 (en) 2001-08-20 2010-06-22 President And Fellows Of Harvard College Fluidic arrays and method of using
US20100173394A1 (en) 2008-09-23 2010-07-08 Colston Jr Billy Wayne Droplet-based assay system
US20100173293A1 (en) 2003-09-19 2010-07-08 Life Technologies Corporation High Density Sequence Detection Methods
US20100184069A1 (en) 2009-01-21 2010-07-22 Streck, Inc. Preservation of fetal nucleic acids in maternal plasma
US20100183504A1 (en) 2007-06-14 2010-07-22 Fanqing Frank Chen Multimodal imaging probes for in vivo targeted and non-targeted imaging and therapeutics
US20100216128A1 (en) 2006-02-07 2010-08-26 Stokes Bio Ltd. Methods for analyzing agricultural and environmental samples
JP2010198393A (en) 2009-02-26 2010-09-09 Alpine Electronics Inc Map display device
US20100233083A1 (en) 2006-03-21 2010-09-16 Dsm Ip Assets B.V. Microparticles comprising a crosslinked polymer
US20100240101A1 (en) 2009-03-19 2010-09-23 Massachusetts Institute Of Technology Parallel Proximity Ligation Event Analysis
US20100248237A1 (en) 2009-02-25 2010-09-30 Thomas Froehlich Miniaturized, high-throughput nucleic acid analysis
JP2010217477A (en) 2009-03-17 2010-09-30 Yamaha Corp Waveform data generating method
WO2010115154A1 (en) 2009-04-02 2010-10-07 Fluidigm Corporation Multi-primer amplification method for barcoding of target nucleic acids
US7814175B1 (en) 2001-05-14 2010-10-12 At&T Intellectual Property Ii, L.P. System having generalized client-server computing
US20100273173A1 (en) 2007-12-26 2010-10-28 Arkray, Inc. Method for amplifying target nucleic acid sequence and probe used for the same
US20100282617A1 (en) 2006-12-14 2010-11-11 Ion Torrent Systems Incorporated Methods and apparatus for detecting molecular interactions using fet arrays
US20100300895A1 (en) 2009-05-29 2010-12-02 Ion Torrent Systems, Inc. Apparatus and methods for performing electrochemical reactions
US20100301398A1 (en) 2009-05-29 2010-12-02 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
US20100304982A1 (en) 2009-05-29 2010-12-02 Ion Torrent Systems, Inc. Scaffolded nucleic acid polymer particles and methods of making and using
US20100300559A1 (en) 2008-10-22 2010-12-02 Ion Torrent Systems, Inc. Fluidics system for sequential delivery of reagents
WO2010151776A2 (en) 2009-06-26 2010-12-29 President And Fellows Of Harvard College Fluid injection
US20110000560A1 (en) 2009-03-23 2011-01-06 Raindance Technologies, Inc. Manipulation of Microfluidic Droplets
WO2011005221A1 (en) 2009-07-07 2011-01-13 Agency For Science, Technology And Research Methods of identifying a pair of binding partners
US20110024455A1 (en) 2004-05-21 2011-02-03 Pepsico, Inc. Beverage dispensing system with a head capable of dispensing plural different beverages
US20110033854A1 (en) 2007-12-05 2011-02-10 Complete Genomics, Inc. Methods and compositions for long fragment read sequencing
US20110045462A1 (en) 2006-11-14 2011-02-24 The Regents Of The University Of California Digital analysis of gene expression
US7897044B2 (en) 2005-03-11 2011-03-01 Centre National De La Recherche Scientifique Fluid separation device
US20110053798A1 (en) 2009-09-02 2011-03-03 Quantalife, Inc. System for mixing fluids by coalescence of multiple emulsions
US20110059556A1 (en) 2009-09-04 2011-03-10 The Research Foundation Of State University Of New York Rapid and Continuous Analyte Processing in Droplet Microfluidic Devices
US20110059435A1 (en) * 2005-10-24 2011-03-10 The John Hopkins University Methods for Beaming
WO2011042564A1 (en) 2009-10-09 2011-04-14 Universite De Strasbourg Labelled silica-based nanomaterial with enhanced properties and uses thereof
US20110104725A1 (en) 2008-05-02 2011-05-05 Advanced Liquid Logic, Inc. Method of Effecting Coagulation in a Droplet
US20110111981A1 (en) 2008-12-04 2011-05-12 Masschusetts Institute Of Technology Method for detecting active and latent virally infected cells
US20110151444A1 (en) 2009-12-18 2011-06-23 Andreas Albers Method for detection of an rna molecule, a kit and use related therefor
WO2011079176A2 (en) 2009-12-23 2011-06-30 Raindance Technologies, Inc. Microfluidic systems and methods for reducing the exchange of molecules between droplets
US20110159499A1 (en) 2009-11-25 2011-06-30 Quantalife, Inc. Methods and compositions for detecting genetic material
US7990525B2 (en) 2005-08-08 2011-08-02 Bay Bioscience Kabushiki Kaisha Flow cytometer and flow cytometry
US20110190146A1 (en) 2008-04-28 2011-08-04 President And Fellows Of Harvard College Microfluidic device for storage and well-defined arrangement of droplets
US20110188717A1 (en) 2008-07-15 2011-08-04 Universite Pierre Et Marie Curie (Paris 6) Method and device for reading an emulsion
WO2011100604A2 (en) 2010-02-12 2011-08-18 Raindance Technologies, Inc. Digital analyte analysis
US20110218123A1 (en) 2008-09-19 2011-09-08 President And Fellows Of Harvard College Creation of libraries of droplets and related species
US20110223314A1 (en) 2010-03-10 2011-09-15 Xiaoxiao Zhang Efficient microencapsulation
US20110257031A1 (en) 2010-02-12 2011-10-20 Life Technologies Corporation Nucleic acid, biomolecule and polymer identifier codes
US20110267457A1 (en) 2007-12-21 2011-11-03 David A Weitz Systems and methods for nucleic acid sequencing
US20110274706A1 (en) 2010-05-04 2011-11-10 General Electric Company Nucleic acid delivery vehicle and uses thereof
US20110275063A1 (en) 2008-07-11 2011-11-10 President And Fellows Of Harvard College Systems and methods of droplet-based selection
US20110311978A1 (en) 2008-09-23 2011-12-22 Quantalife, Inc. System for detection of spaced droplets
US20120010085A1 (en) 2010-01-19 2012-01-12 Rava Richard P Methods for determining fraction of fetal nucleic acids in maternal samples
US20120014977A1 (en) 2010-02-19 2012-01-19 National University Corporation Chiba University NOVEL ALTERNATIVE SPLICING VARIANT OF OATP1B3 mRNA
US20120015822A1 (en) 2008-12-19 2012-01-19 President And Fellows Of Harvard College Particle-assisted nucleic acid sequencing
US20120021919A1 (en) 2010-07-23 2012-01-26 Thomas Scholl Identification of Differentially Represented Fetal or Maternal Genomic Regions and Uses Thereof
US20120021930A1 (en) 2005-11-22 2012-01-26 Stichting Dienst Landbouwkundig Onderzoek Multiplex Nucleic Acid Detection
WO2012022976A1 (en) 2010-08-20 2012-02-23 Sphere Fluidics Limited Method of providing a chemical or biological material in quantised form and system therefor
WO2012036679A1 (en) 2010-09-15 2012-03-22 Life Technologies Corporation Methods and apparatus for measuring analytes
WO2012045012A2 (en) 2010-09-30 2012-04-05 Raindance Technologies, Inc. Sandwich assays in droplets
WO2012048341A1 (en) 2010-10-08 2012-04-12 President And Fellows Of Harvard College High-throughput single cell barcoding
US20120088691A1 (en) 2010-10-08 2012-04-12 Gao Chen Highly multiplexed real-time pcr using encoded microbeads
WO2012083225A2 (en) 2010-12-16 2012-06-21 Gigagen, Inc. System and methods for massively parallel analysis of nycleic acids in single cells
US20120164652A1 (en) 2010-12-27 2012-06-28 Ibis Biosciences, Inc. Quantitating high titer samples by digital pcr
US20120167142A1 (en) 2010-12-23 2012-06-28 Eldon Technology Limited Methods and apparatuses to facilitate preselection of programming preferences
US20120165219A1 (en) 2009-09-01 2012-06-28 Koninklijke Philips Electronics N.V. Devices and methods for microarray selection
US20120171667A1 (en) 2006-06-14 2012-07-05 Daniel Shoemaker Rare Cell Analysis Using Sample Splitting And DNA Tags
US20120190032A1 (en) 2010-03-25 2012-07-26 Ness Kevin D Droplet generation for droplet-based assays
WO2012103339A1 (en) 2011-01-26 2012-08-02 Firestone Industrial Products Company, Llc Gas spring piston as well as gas spring assembly, suspension system, kit and method including same
WO2012106385A2 (en) 2011-01-31 2012-08-09 Apprise Bio, Inc. Methods of identifying multiple epitopes in cells
US20120208241A1 (en) 2011-02-11 2012-08-16 Raindance Technologies, Inc. Thermocycling device for nucleic acid amplification and methods of use
US20120208705A1 (en) 2011-02-10 2012-08-16 Steemers Frank J Linking sequence reads using paired code tags
US20120220494A1 (en) 2011-02-18 2012-08-30 Raindance Technolgies, Inc. Compositions and methods for molecular labeling
US20120219947A1 (en) 2011-02-11 2012-08-30 Raindance Technologies, Inc. Methods for forming mixed droplets
US20120231972A1 (en) 2009-03-19 2012-09-13 Golyshin Peter N Probe Compound for Detecting and Isolating Enzymes and Means and Methods Using the Same
US20120244043A1 (en) 2011-01-28 2012-09-27 Sean Leblanc Elastomeric gasket for fluid interface to a microfluidic chip
US8278711B2 (en) 2010-11-23 2012-10-02 General Electric Company Semiconductor device and method of making the same
US20120252012A1 (en) 2009-05-14 2012-10-04 Assistance Publique - Hopitaux De Marseille Method for detecting prokaryotic dna from a feces sample
US20120253689A1 (en) 2005-06-07 2012-10-04 Rogan Peter K Ab initio generation of single copy genomic probes
US20120258516A1 (en) 2011-04-07 2012-10-11 Life Technologies Corporation System and Methods for Making and Processing Emulsions
WO2012142213A2 (en) 2011-04-15 2012-10-18 The Johns Hopkins University Safe sequencing system
US20120264646A1 (en) 2008-07-18 2012-10-18 Raindance Technologies, Inc. Enzyme quantification
JP2012204765A (en) 2011-03-28 2012-10-22 Tamura Seisakusho Co Ltd Reflow device
US20120288857A1 (en) 2011-02-03 2012-11-15 Fluidigm Corporation Multifunctional probe-primers
US8318434B2 (en) 2007-09-21 2012-11-27 Katholieke Universiteit Leuven, K.U.Leuven R&D Method for introducing a sample specific DNA tag into a plurality of DNA fragments from a plurality of samples
US20120302448A1 (en) 2010-02-12 2012-11-29 Raindance Technologies, Inc. Digital analyte analysis
US20120309002A1 (en) 2011-06-02 2012-12-06 Raindance Technologies, Inc. Sample multiplexing
US20120322058A1 (en) 2011-02-09 2012-12-20 Bio-Rad Laboratories Analysis of nucleic acids
WO2013014356A2 (en) 2011-07-28 2013-01-31 Valeo Equipements Electriques Moteur Motor vehicle starter circuit comprising a voltage raising device and starter so equipped
US8383061B2 (en) 2005-05-02 2013-02-26 Massachusetts Institute Of Technology Microfluidic bubble logic devices
US20130099018A1 (en) 2011-07-20 2013-04-25 Raindance Technolgies, Inc. Manipulating droplet size
US20130109577A1 (en) 2011-10-14 2013-05-02 Pacific Biosciences Of California, Inc. Real-time redox sequencing
US8436993B2 (en) 2007-04-02 2013-05-07 Life Technologies Corporation Methods and systems for controlling the flow of particles for detection
US20130123339A1 (en) 2010-05-12 2013-05-16 Protiva Biotherapeutics Inc Compositions and methods for silencing apolipoprotein b
US20130143745A1 (en) 2011-06-24 2013-06-06 QB3/Pharm-Chem Digital Health Garage Compositions and methods for identifying the essential genome of an organism
US8462269B2 (en) 2007-11-16 2013-06-11 Mediatek Inc. Devices and methods for extracting a synchronization signal from a video signal
US20130157870A1 (en) 2011-09-09 2013-06-20 The Board Of Trustees Of The Leland Stanford Junior University Methods for obtaining a sequence
US20130178378A1 (en) 2011-06-09 2013-07-11 Andrew C. Hatch Multiplex digital pcr
US20130203606A1 (en) 2010-02-25 2013-08-08 Advanced Liquid Logic Inc Method of Preparing a Nucleic Acid Library
US20130210638A1 (en) 2012-02-10 2013-08-15 Jeffrey Charles Olson Methods for sequencing nucleic acid
WO2013120089A1 (en) 2012-02-10 2013-08-15 Raindance Technologies, Inc. Molecular diagnostic screening assay
US20130217071A1 (en) 2011-12-30 2013-08-22 Luz Montesclaros Methods and compositions for performing nucleic acid amplification reactions
US20130225418A1 (en) 2012-02-24 2013-08-29 Andrew Watson Labeling and sample preparation for sequencing
US20130225623A1 (en) 2010-10-27 2013-08-29 Mount Sinai School Of Medicine Methods of Treating Psychiatric or Neurological Disorders with MGLUR Antagonists
US20130244906A1 (en) 2011-05-05 2013-09-19 Biopico Systems Microfluidic devices and methods based on massively parallel picoreactors for cell and molecular diagnostics
US20130260447A1 (en) 2006-05-11 2013-10-03 Darren R. Link Systems and methods for handling microfluidic droplets
US20130288254A1 (en) 2009-08-13 2013-10-31 Advanced Liquid Logic, Inc. Droplet Actuator and Droplet-Based Techniques
US20130295568A1 (en) 2010-02-12 2013-11-07 Darren Roy Link Digital analyte analysis
US20130295567A1 (en) 2010-02-12 2013-11-07 Darren Roy Link Digital analyte analysis
US20130296535A1 (en) 2010-10-08 2013-11-07 President And Fellows Of Harvard College High-Throughput Immune Sequencing
WO2013165748A1 (en) 2012-04-30 2013-11-07 Raindance Technologies, Inc Digital analyte analysis
WO2014026031A1 (en) 2012-08-10 2014-02-13 Sequenta, Inc. High sensitivity mutation detection using sequence tags
US20140045712A1 (en) 2010-02-12 2014-02-13 Darren Roy Link Digital analyte analysis
US20140065631A1 (en) * 2012-08-31 2014-03-06 Roche Molecular Systems, Inc. Microfluidic Chip, Device and System for the Generation of Aqueous Droplets in Emulsion Oil for Nucleic Acid Amplification
US8673595B2 (en) 2009-06-29 2014-03-18 Kabushiki Kaisha Toshiba Sample analysis method and assay kit used therein
US20140113300A1 (en) 2012-10-22 2014-04-24 Raindance Technologies, Inc. Methods for analyzing dna
WO2014065756A1 (en) 2012-10-22 2014-05-01 National University Of Singapore Assay for the parallel detection of biological material based on pcr
US8715934B2 (en) 2006-06-19 2014-05-06 The Johns Hopkins University Single-molecule PCR on microparticles in water-in-oil emulsions
US20140155274A1 (en) 2011-03-24 2014-06-05 President And Fellows Of Harvard College Single Cell Nucleic Acid Detection and Analysis
US20140256585A1 (en) 2013-03-07 2014-09-11 Bio-Rad Laboratories, Inc. Repetitive reverse transcription partition assay
US20140274786A1 (en) 2013-03-15 2014-09-18 Bio-Rad Laboratories, Inc. Digital assays with associated targets
WO2014165559A2 (en) 2013-04-02 2014-10-09 Raindance Technologies, Inc. Systems and methods for handling microfluidic droplets
WO2014194131A2 (en) 2013-05-29 2014-12-04 Gnubio, Inc. Systems and methods for sequencing in emulsion based microfluidics
WO2014204939A2 (en) 2013-06-17 2014-12-24 Kim Lewis Methods for quantitative determination of protein-nucleic acid interactions in complex mixtures
US20150018236A1 (en) 2012-02-02 2015-01-15 Invenra Inc. High throughput screen for biologically active polypeptides
WO2015013681A1 (en) 2013-07-25 2015-01-29 Bio-Rad Laboratories, Inc. Genetic assays
US20150099266A1 (en) 2013-10-04 2015-04-09 Raindance Technologies, Inc. Digital analysis of nucleic acid modification
US20150167066A1 (en) 2013-12-12 2015-06-18 Raindance Technologies, Inc. Distinguishing rare variations in a nucleic acid sequence from a sample
US20150184256A1 (en) 2013-12-31 2015-07-02 Raindance Technologies, Inc. System and method for detection of rna species
US9080056B2 (en) 2008-11-26 2015-07-14 University College Cork—National University of Ireland, Cork Process for preparing silica microparticles
US20150197790A1 (en) 2014-01-10 2015-07-16 Bio-Rad Laboratories, Inc. Intercalating dyes for differential detection
US20150247191A1 (en) 2012-09-12 2015-09-03 The Regents Of The University Of California Accurate genome sequencing of single cells by single-stranded amplification and sequencing
US20150258520A1 (en) 2012-11-30 2015-09-17 The Broad Institute Inc. High-throughput dynamic reagent delivery system
WO2015164212A1 (en) 2014-04-21 2015-10-29 President And Fellows Of Harvard College Systems and methods for barcoding nucleic acids
WO2015200541A1 (en) 2014-06-24 2015-12-30 Bio-Rad Laboratories, Inc. Digital pcr barcoding
WO2015200893A2 (en) 2014-06-26 2015-12-30 10X Genomics, Inc. Methods of analyzing nucleic acids from individual cells or cell populations
US9273349B2 (en) 2013-03-14 2016-03-01 Affymetrix, Inc. Detection of nucleic acids
US9322511B2 (en) 2006-02-07 2016-04-26 Stokes Bio Limited Liquid bridge and system
US20160200847A1 (en) 2014-12-12 2016-07-14 Marcella Chiari Clickable polymers and gels for microarray and other applications
US20160304954A1 (en) 2013-12-11 2016-10-20 Accuragen, Inc. Compositions and methods for detecting rare sequence variants
US9592510B2 (en) 2006-06-30 2017-03-14 Canon U.S. Life Sciences, Inc. Real-time PCR in micro-channels
WO2017100350A1 (en) 2015-12-07 2017-06-15 Raindance Technologies, Inc. Multiplexing in partitions using microparticles
WO2017117358A1 (en) 2015-12-30 2017-07-06 Bio-Rad Laboratories, Inc. Digital protein quantification
US9857202B2 (en) 2015-05-28 2018-01-02 Sanyo Denki Co., Ltd. Sensor for motor
US20180057868A1 (en) 2016-08-30 2018-03-01 Integrated Dna Technologies, Inc. Cleavable hairpin primers
US20180304222A1 (en) 2014-04-21 2018-10-25 President And Fellows Of Harvard College Systems and methods for barcoding nucleic acids
US10526605B2 (en) 2016-06-03 2020-01-07 Purdue Research Foundation siRNA compositions that specifically downregulate expression of a variant of the PNPLA3 gene and methods of use thereof for treating a chronic liver disease or alcoholic liver disease (ALD)
US10626451B2 (en) 2008-08-12 2020-04-21 Stokes Bio Limited Methods and devices for digital PCR
US10647981B1 (en) 2015-09-08 2020-05-12 Bio-Rad Laboratories, Inc. Nucleic acid library generation methods and compositions
US20220042995A1 (en) 2008-07-18 2022-02-10 Bio-Rad Laboratories, Inc. Enzyme quantification

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05265447A (en) 1992-03-18 1993-10-15 Kawai Musical Instr Mfg Co Ltd Electronic musical instrument capable of deciding finger of hand operating keyboard
JP3232525B2 (en) 1995-08-22 2001-11-26 信越化学工業株式会社 Water repellent agent
IT1309792B1 (en) * 1999-04-22 2002-01-30 Eltek Spa HOUSEHOLD APPLIANCES USING WATER, IN PARTICULAR A WASHING MACHINE, WITH PERFECTED DEVICE FOR BLAST CHILLING
JP2002372016A (en) 2001-06-18 2002-12-26 Mitsubishi Heavy Ind Ltd Bolt connection structure
US6598397B2 (en) * 2001-08-10 2003-07-29 Energetix Micropower Limited Integrated micro combined heat and power system
FR2844039B1 (en) 2002-09-04 2005-04-29 Air Liquide PROCESS AND PLANT FOR PRODUCING OXYGEN AND RARE GASES BY CRYOGENIC AIR DISTILLATION
AU2003291144A1 (en) * 2002-11-21 2004-06-18 Primera Biosystems Sampling method and apparatus for amplification reaction analysis
JP4752047B2 (en) * 2004-06-30 2011-08-17 国立大学法人北海道大学 Method for producing cell culture substrate and cell culture method
US7079415B2 (en) 2004-06-30 2006-07-18 Stmicroelectronics, Inc. Magnetic random access memory element
US20060021280A1 (en) 2004-07-30 2006-02-02 Hamilton Daniel B Reformer, and methods of making and using the same
US7604940B1 (en) * 2005-03-16 2009-10-20 Applied Biosystems, Llc Compositions and methods for analyzing isolated polynucleotides

Patent Citations (1509)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2097692A (en) 1936-03-23 1937-11-02 Bohn Aluminium & Brass Corp Method and machine for forming bearing shells
US2164172A (en) 1938-04-30 1939-06-27 Gen Electric Liquid-dispensing apparatus
US2636855A (en) 1948-03-25 1953-04-28 Hilger & Watts Ltd Method of producing photoconductive coatings
US2656508A (en) 1949-08-27 1953-10-20 Wallace H Coulter Means for counting particles suspended in a fluid
US2692800A (en) 1951-10-08 1954-10-26 Gen Electric Nozzle flow control
US2797149A (en) 1953-01-08 1957-06-25 Technicon International Ltd Methods of and apparatus for analyzing liquids containing crystalloid and non-crystalloid constituents
US2879141A (en) 1955-11-16 1959-03-24 Technicon Instr Automatic analyzing apparatus
US2971700A (en) 1957-07-22 1961-02-14 Vilbiss Co Apparatus for coating articles with chemically reactive liquids
US3608821A (en) 1965-10-15 1971-09-28 Agfa Gevaert Ag Electrostatic atomization of liquids
GB1148543A (en) 1966-01-10 1969-04-16 Bachofen Willy A Sight glass fitting for optical flow supervision
US3479141A (en) 1967-05-17 1969-11-18 Technicon Corp Method and apparatus for analysis
US3980541A (en) 1967-06-05 1976-09-14 Aine Harry E Electrode structures for electric treatment of fluids and filters using same
US3621059A (en) 1969-07-30 1971-11-16 Du Pont Amides of hexafluoropropylene oxide polymer acids and polyalklene oxide
US3784471A (en) 1970-05-11 1974-01-08 Avco Corp Solid additives dispersed in perfluorinated liquids with perfluoroalkyl ether dispersants
US3828085A (en) 1970-07-09 1974-08-06 Allied Chem Novel amidoamine oxides
DE2100685A1 (en) 1971-01-08 1972-07-20 Badische Anilin- & Soda-Fabrik Ag, 6700 Ludwigshafen Pure 4-amino-5-halo-6-pyridazones sepn - from isomers by extraction with halohydrocarbons, alkanols or ethers
US3698635A (en) 1971-02-22 1972-10-17 Ransburg Electro Coating Corp Spray charging device
US3816331A (en) 1972-07-05 1974-06-11 Ncr Continuous encapsulation and device therefor
CH563087A5 (en) 1972-10-06 1975-06-13 Westinghouse Electric Corp
CH563807A5 (en) 1973-02-14 1975-07-15 Battelle Memorial Institute Fine granules and microcapsules mfrd. from liquid droplets - partic. of high viscosity requiring forced sepn. of droplets
GB1446998A (en) 1974-02-25 1976-08-18 Sauter Ag Apparatus for mixing at least two fluent media
US3930061A (en) 1974-04-08 1975-12-30 Ransburg Corp Electrostatic method for forming structures and articles
US4059552A (en) 1974-06-21 1977-11-22 The Dow Chemical Company Cross-linked water-swellable polymer particles
US3960187A (en) 1974-07-23 1976-06-01 Usm Corporation Method and device for metering and dispersing fluid materials
US3982541A (en) 1974-07-29 1976-09-28 Esperance Jr Francis A L Eye surgical instrument
US4022575A (en) 1974-09-16 1977-05-10 Block Engineering, Inc. Automatic chemical analyzer
US4297345A (en) 1975-04-14 1981-10-27 Beecham Group Limited Antibacterial agents
US4034966A (en) 1975-11-05 1977-07-12 Massachusetts Institute Of Technology Method and apparatus for mixing particles
US4014469A (en) 1975-11-17 1977-03-29 Kozo Sato Nozzle of gas cutting torch
JPS5372016A (en) 1976-12-08 1978-06-27 Toyo Tire & Rubber Co Ltd Apparatus for preparation and supply of heavy oil w/o emulsion fuel
US4117550A (en) 1977-02-14 1978-09-26 Folland Enertec Ltd. Emulsifying system
US4091042A (en) 1977-08-19 1978-05-23 American Cyanamid Company Continuous adiabatic process for the mononitration of benzene
US4130394A (en) 1977-10-03 1978-12-19 Technicon Instruments Corporation Short sample detection
GB2005224A (en) 1977-10-03 1979-04-19 Technicon Instr Method and apparatus for dispensing liquids
FR2404834A1 (en) 1977-10-03 1979-04-27 Technicon Instr LIQUID SAMPLES PROBE WITH MOVABLE MARK INDICATING ASPIRE VOLUMES
JPS5455495A (en) 1977-10-03 1979-05-02 Technicon Instr Method of measuring liquid absorption and device for diluting liquid
AU4032078A (en) 1977-10-03 1980-04-03 Technicon Instr Short sample detection
CA1093344A (en) 1977-10-03 1981-01-13 Kent M. Negersmith Short sample detection
US4962885A (en) 1978-04-17 1990-10-16 Coffee Ronald A Process and apparatus for spraying liquid
US4378957A (en) 1978-08-11 1983-04-05 Malkin Daniel D Reduction gear of electronic wristwatch with stepping motor and sweep second hand
GB2047880A (en) 1979-03-16 1980-12-03 Technicon Instr Determination of characteristics of segmented fluid
FR2451579A1 (en) 1979-03-16 1980-10-10 Technicon Instr METHOD AND APPARATUS FOR EXTERIOR DETERMINATION OF THE CHARACTERISTICS OF A SEGMENTED FLUID
JPS55125472A (en) 1979-03-16 1980-09-27 Technicon Instr Method and device for detecting fluid flow section and section boundary and method and device for determining liquid section volume
US4210809A (en) 1979-03-16 1980-07-01 Technicon Instruments Corporation Method and apparatus for the non-invasive determination of the characteristics of a segmented fluid stream
US4279345A (en) 1979-08-03 1981-07-21 Allred John C High speed particle sorter using a field emission electrode
US4315754A (en) 1979-08-28 1982-02-16 Bifok Ab Flow injection analysis with intermittent flow
GB2062225A (en) 1979-08-28 1981-05-20 Bifok Ab Continuous flow analysis system with intermittent injection
FR2469714A1 (en) 1979-08-28 1981-05-22 Bifok Ab METHOD OF ANALYSIS BY INJECTION IN A FLOWING FLUID, WITH INTERMITTENT FLOW OF THE FLUID
JPS5636053A (en) 1979-08-28 1981-04-09 Bifok Ab Method of continuous flowing analysis
US4266721A (en) 1979-09-17 1981-05-12 Ppg Industries, Inc. Spray application of coating compositions utilizing induction and corona charging means
US4383767A (en) 1979-11-05 1983-05-17 Agency Of Industrial Science & Technology Method for blending by combining fine particles
AU6415380A (en) 1979-11-21 1981-05-28 Technicon Instruments Corportion Sample stream apparatus
US4253846A (en) 1979-11-21 1981-03-03 Technicon Instruments Corporation Method and apparatus for automated analysis of fluid samples
JPS56124052A (en) 1979-11-21 1981-09-29 Technicon Instr Quantitatively measuring method and device and continuous analyzer
DE3042915A1 (en) 1979-11-21 1981-09-03 Technicon Instruments Corp., Tarrytown, N.Y. METHOD AND DEVICE FOR THE AUTOMATIC ANALYSIS OF FLUID SAMPLES
GB2064114A (en) 1979-11-21 1981-06-10 Technicon Instr Continuous flow analysis method and apparatus
FR2470385A1 (en) 1979-11-21 1981-05-29 Technicon Instr SYSTEM, METHOD AND APPARATUS FOR AUTOMATIC ANALYSIS OF FLUID SAMPLES
EP0047130B1 (en) 1980-08-28 1985-02-13 E.I. Du Pont De Nemours And Company Flow analysis
WO1984002000A1 (en) 1981-01-10 1984-05-24 Shaw Stewart P D Chemical droplet reactor
AU1045983A (en) 1981-01-10 1984-06-04 Shaw, Stewart P D Chemical droplet reactor
GB2097692A (en) 1981-01-10 1982-11-10 Shaw Stewart P D Combining chemical reagents
BR8200642A (en) 1981-02-06 1982-12-14 Honda Motor Co Ltd APPLIANCE FOR WELDING
US4508265A (en) 1981-06-18 1985-04-02 Agency Of Industrial Science & Technology Method for spray combination of liquids and apparatus therefor
US4439980A (en) 1981-11-16 1984-04-03 The United States Of America As Represented By The Secretary Of The Navy Electrohydrodynamic (EHD) control of fuel injection in gas turbines
US4996004A (en) 1982-08-14 1991-02-26 Bayer Aktiengesellschaft Preparation of pharmaceutical or cosmetic dispersions
JPS5949832A (en) 1982-08-14 1984-03-22 バイエル・アクチエンゲゼルシヤフト Dispersion manufacturing method and device
US4779805A (en) 1982-10-13 1988-10-25 Imperial Chemical Industries Plc Electrostatic sprayhead assembly
JPS59102163A (en) 1982-11-15 1984-06-13 テクニコン，インストルメンツ，コ−ポレ−シヨン Continuous flowing method, continuous flowing device and single flow-path continuous flow analyzer
US4853336A (en) 1982-11-15 1989-08-01 Technicon Instruments Corporation Single channel continuous flow system
US4533634A (en) 1983-01-26 1985-08-06 Amf Inc. Tissue culture medium
US4585209A (en) 1983-10-27 1986-04-29 Harry E. Aine Miniature valve and method of making same
US4618476A (en) 1984-02-10 1986-10-21 Eastman Kodak Company Capillary transport device having speed and meniscus control means
US4566908A (en) 1984-02-24 1986-01-28 Mita Industrial Company, Limited Azoic pigments having a silica core
US4865444A (en) 1984-04-05 1989-09-12 Mobil Oil Corporation Apparatus and method for determining luminosity of hydrocarbon fuels
US4675285A (en) 1984-09-19 1987-06-23 Genetics Institute, Inc. Method for identification and isolation of DNA encoding a desired protein
US4883750A (en) 1984-12-13 1989-11-28 Applied Biosystems, Inc. Detection of specific sequences in nucleic acids
US4801086A (en) 1985-02-19 1989-01-31 Imperial Chemical Industries Plc Spraying apparatus
US4859363A (en) 1985-02-26 1989-08-22 I.S.C. Chemicals Limited Emulsions of perfluorocarbons in aqueous media
US4676274A (en) 1985-02-28 1987-06-30 Brown James F Capillary flow control
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US5656493A (en) 1985-03-28 1997-08-12 The Perkin-Elmer Corporation System for automated performance of the polymerase chain reaction
US4683202B1 (en) 1985-03-28 1990-11-27 Cetus Corp
US4739044A (en) 1985-06-13 1988-04-19 Amgen Method for derivitization of polynucleotides
US4801529A (en) 1985-06-18 1989-01-31 Brandeis University Methods for isolating mutant microoganisms using microcapsules coated with indicator material
US4963498A (en) 1985-08-05 1990-10-16 Biotrack Capillary flow device
US4757141A (en) 1985-08-26 1988-07-12 Applied Biosystems, Incorporated Amino-derivatized phosphite and phosphate linking agents, phosphoramidite precursors, and useful conjugates thereof
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683195B1 (en) 1986-01-30 1990-11-27 Cetus Corp
US4795330A (en) 1986-02-21 1989-01-03 Imperial Chemical Industries Plc Apparatus for particles
US4829996A (en) 1986-02-21 1989-05-16 Imperial Chemical Industries Plc Apparatus for producing a spray of droplets of a liquid
US5333675A (en) 1986-02-25 1994-08-02 Hoffmann-La Roche Inc. Apparatus and method for performing automated amplification of nucleic acid sequences and assays using heating and cooling steps
US5333675C1 (en) 1986-02-25 2001-05-01 Perkin Elmer Corp Apparatus and method for performing automated amplification of nucleic acid sequences and assays using heating and cooling steps
US5468613A (en) 1986-03-13 1995-11-21 Hoffmann-La Roche Inc. Process for detecting specific nucleotide variations and genetic polymorphisms present in nucleic acids
EP0249007A3 (en) 1986-04-14 1991-03-20 The General Hospital Corporation A method of screening hybridomas
EP0249007A2 (en) 1986-04-14 1987-12-16 The General Hospital Corporation A method of screening hybridomas
US5204112A (en) 1986-06-16 1993-04-20 The Liposome Company, Inc. Induction of asymmetry in vesicles
US4767929A (en) 1986-10-06 1988-08-30 The United States Of America As Represented By The United State Department Of Energy Extended range radiation dose-rate monitor
US4767515A (en) 1987-07-30 1988-08-30 The United States Of America As Represented By The United States Department Of Energy Surface area generation and droplet size control in solvent extraction systems utilizing high intensity electric fields
US5149625A (en) 1987-08-11 1992-09-22 President And Fellows Of Harvard College Multiplex analysis of DNA
US5399461A (en) 1987-08-21 1995-03-21 Sharp Kabushiki Kaisha Optical disk for use in optical memory devices
GB2210532A (en) 1987-09-30 1989-06-07 Sony Corp Digital signal processing circuit for video camera
US4931225A (en) 1987-12-30 1990-06-05 Union Carbide Industrial Gases Technology Corporation Method and apparatus for dispersing a gas into a liquid
US5180662A (en) 1988-01-05 1993-01-19 The United States Of America As Represented By The Department Of Health And Human Services Cytotoxic T lymphocyte activation assay
US4856363A (en) 1988-02-10 1989-08-15 Wickes Manufacturing Company Parking brake assembly
US5185099A (en) 1988-04-20 1993-02-09 Institut National De Recherche Chimique Appliquee Visco-elastic, isotropic materials based on water, fluorinate sufactants and fluorinated oils, process for their preparation, and their use in various fields, such as optics, pharmacology and electrodynamics
US5055390A (en) 1988-04-22 1991-10-08 Massachusetts Institute Of Technology Process for chemical manipulation of non-aqueous surrounded microdroplets
US4908112A (en) 1988-06-16 1990-03-13 E. I. Du Pont De Nemours & Co. Silicon semiconductor wafer for analyzing micronic biological samples
US5498523A (en) 1988-07-12 1996-03-12 President And Fellows Of Harvard College DNA sequencing with pyrophosphatase
US5096615A (en) 1988-07-19 1992-03-17 The United States Of America As Represented By The United States Department Of Energy Solid aerosol generator
US5104813A (en) 1989-04-13 1992-04-14 Biotrack, Inc. Dilution and mixing cartridge
US4981580A (en) 1989-05-01 1991-01-01 Coulter Corporation Coincidence arbitration in a flow cytomery sorting system
US5188291A (en) 1989-05-31 1993-02-23 Her Majesty The Queen In Right Of New Zealand Fluid distribution device
US5445934A (en) 1989-06-07 1995-08-29 Affymax Technologies N.V. Array of oligonucleotides on a solid substrate
EP0402995A2 (en) 1989-06-12 1990-12-19 Johnson &amp; Johnson Clinical Diagnostics, Inc. Temperature control device and reaction vessel
WO1990015807A1 (en) 1989-06-22 1990-12-27 Applications Et Transferts De Technologies Avancées Fluorine and phosphorous-containing amphiphilic molecules with surfactant properties
US5399491A (en) 1989-07-11 1995-03-21 Gen-Probe Incorporated Nucleic acid sequence amplification methods
FR2650657A1 (en) 1989-08-05 1991-02-08 Scras Sa APPARATUS FOR THE AUTOMATIC AND REPEATED EXECUTION OF A THERMAL CYCLE FOR THE TREATMENT OF BIOLOGICAL SAMPLES
US5192659A (en) 1989-08-25 1993-03-09 Genetype Ag Intron sequence analysis method for detection of adjacent and remote locus alleles as haplotypes
EP0418635A1 (en) 1989-09-12 1991-03-27 Biotest AG Enzyme immunoassay using peroxidase as the marker enzyme
WO1991005058A1 (en) 1989-10-05 1991-04-18 Glenn Kawasaki Cell-free synthesis and isolation of novel genes and polypeptides
US5310653A (en) 1989-10-24 1994-05-10 Board Of Regents, The University Of Texas System Tumor marker protein and antibodies thereto for cancer risk assessment or diagnosis
US5378957A (en) 1989-11-17 1995-01-03 Charged Injection Corporation Methods and apparatus for dispersing a fluent material utilizing an electron beam
WO1991007772A1 (en) 1989-11-17 1991-05-30 Charged Injection Corporation Methods and apparatus for dispersing a fluent material utilizing an electron beam
US5207973A (en) 1989-11-27 1993-05-04 Martin Marietta Energy Systems, Inc. Method and apparatus for the production of metal oxide powder
US4941959A (en) 1989-11-27 1990-07-17 Martin Marietta Energy Systems, Inc. Electric field-driven, magnetically-stabilized ferro-emulsion phase contactor
US5122360A (en) 1989-11-27 1992-06-16 Martin Marietta Energy Systems, Inc. Method and apparatus for the production of metal oxide powder
US5313009A (en) 1990-01-04 1994-05-17 Nrm International Technologies C.V. Nitration process
US5091652A (en) 1990-01-12 1992-02-25 The Regents Of The University Of California Laser excited confocal microscope fluorescence scanner and method
JPH03232525A (en) 1990-02-07 1991-10-16 Gunma Univ Formation of uniform liquid drop
US5188290A (en) 1990-02-16 1993-02-23 J. Wagner Gmbh Electrostatic compressed air paint spray gun
US5523162A (en) 1990-04-03 1996-06-04 Ppg Industries, Inc. Water repellent surface treatment for plastic and coated plastic substrates
WO1991016966A1 (en) 1990-05-10 1991-11-14 Pharmacia Biosensor Ab Microfluidic structure and process for its manufacture
US5376252A (en) 1990-05-10 1994-12-27 Pharmacia Biosensor Ab Microfluidic structure and process for its manufacture
US5475096A (en) 1990-06-11 1995-12-12 University Research Corporation Nucleic acid ligands
US5270163A (en) 1990-06-11 1993-12-14 University Research Corporation Methods for identifying nucleic acid ligands
US5858670A (en) 1990-07-02 1999-01-12 The Arizona Board Of Regents Bio-oligomer libraries and a method of use thereof
WO1992003734A1 (en) 1990-08-20 1992-03-05 Alain De Weck A method for measuring t-lymphocyte responses by chemiluminescent assays
EP0476178A1 (en) 1990-09-21 1992-03-25 Bioplex Medical B.V. Device for placing styptic material on perforated blood vessels
US6149789A (en) 1990-10-31 2000-11-21 Fraunhofer Gesellschaft Zur Forderung Der Angewandten Forschung E.V. Process for manipulating microscopic, dielectric particles and a device therefor
DE69126763T2 (en) 1990-11-13 1998-02-05 Rhone Poulenc Chimie Process for the preparation of mixed oxalates of ammonium and rare earths and their use for the production of rare earth oxides
US5662874A (en) 1990-11-13 1997-09-02 Rhone-Poulenc Chimie Preparation of ammonium rare earth double oxalates and rare earth oxides produced therefrom
EP0486351B1 (en) 1990-11-13 1997-07-09 Rhone-Poulenc Chimie Method for the production of mixed ammonium-rare earth oxalates and their application to the production of rare earth oxides
FR2669028A1 (en) 1990-11-13 1992-05-15 Rhone Poulenc Chimie PROCESS FOR THE PRODUCTION OF RARE EARTH DUAL OXALATES AND AMMONIUM AND THEIR USES FOR THE MANUFACTURE OF RARE EARTH OXIDES.
US5475610A (en) 1990-11-29 1995-12-12 The Perkin-Elmer Corporation Thermal cycler for automatic performance of the polymerase chain reaction with close temperature control
US5602756A (en) 1990-11-29 1997-02-11 The Perkin-Elmer Corporation Thermal cycler for automatic performance of the polymerase chain reaction with close temperature control
US6263222B1 (en) 1991-03-07 2001-07-17 Masimo Corporation Signal processing apparatus
US5858655A (en) 1991-03-11 1999-01-12 The General Hospital Corporation Method for diagnosing neoplasia by detecting expression of PRAD1 cyclin
US5262027A (en) 1991-03-22 1993-11-16 Martin Marietta Energy Systems, Inc. Method of using an electric field controlled emulsion phase contactor
US5518709A (en) 1991-04-10 1996-05-21 Andaris Limited Preparation of diagnostic agents
US5460945A (en) 1991-05-30 1995-10-24 Center For Blood Research, Inc. Device and method for analysis of blood components and identifying inhibitors and promoters of the inflammatory response
AU2177292A (en) 1991-05-30 1993-01-08 Center For Blood Research, Inc., The Device and method for the analysis of rolling blood leukocytes
NZ264353A (en) 1991-05-30 1996-05-28 For Blood Research Inc Centre Method of collecting or purifying leukocytes from a fluid sample, apparatus, immune response inhibitor test
WO1992021746A1 (en) 1991-05-30 1992-12-10 Center For Blood Research, Inc. Device and method for the analysis of rolling blood leukocytes and identifying inhibitors and promoters
US5452878A (en) 1991-06-18 1995-09-26 Danfoss A/S Miniature actuating device
EP0546174A1 (en) 1991-06-29 1993-06-16 Miyazaki-Ken Monodisperse single and double emulsions and production thereof
US5260466A (en) 1991-08-08 1993-11-09 Tioxide Specialties Limited Preparation of titanium derivatives
ES2095413T3 (en) 1991-08-08 1997-02-16 Tioxide Specialties Ltd PREPARATION OF TITANIUM DERIVATIVES.
EP0528580B1 (en) 1991-08-08 1996-12-04 Tioxide Specialties Limited Preparation of titanium derivatives
IE922432A1 (en) 1991-08-08 1993-02-10 Tioxide Specialties Ltd Preparation of Titanium Derivatives
WO1993003151A1 (en) 1991-08-10 1993-02-18 Medical Research Council Treatment of cell populations
US5830663A (en) 1991-08-10 1998-11-03 Medical Research Council In situ recombinant PCR within single cells
US5454472A (en) 1991-08-19 1995-10-03 Fraunhofer Gesellschaft Zur Forderung Der Angewandten Forschung E.V. Method of continuously separating mixtures of microscopic dielectric particles and apparatus for carrying through this method
EP0718038A2 (en) 1991-08-19 1996-06-26 Fraunhofer-Gesellschaft Zur Förderung Der Angewandten Forschung E.V. Apparatus for separating mixtures of microscopic small dielectric particles dispersed in a fluid or a gel
US5516635A (en) 1991-10-15 1996-05-14 Ekins; Roger P. Binding assay employing labelled reagent
WO1993008278A1 (en) 1991-10-16 1993-04-29 Affymax Technologies N.V. Peptide library and screening method
EP0540281B1 (en) 1991-10-29 1996-07-03 Xerox Corporation Method and apparatus for fabricating bichromal balls for a twisting ball display
US5344594A (en) 1991-10-29 1994-09-06 Xerox Corporation Method for the fabrication of multicolored balls for a twisting ball display
US6048690A (en) 1991-11-07 2000-04-11 Nanogen, Inc. Methods for electronic fluorescent perturbation for analysis and electronic perturbation catalysis for synthesis
US5344489A (en) 1991-11-15 1994-09-06 Manfred R. Kuehnle Synthetic, monodispersed color pigments for the coloration of media such as printing inks, and method and apparatus for making same
US5612188A (en) 1991-11-25 1997-03-18 Cornell Research Foundation, Inc. Automated, multicompartmental cell culture system
US5354670A (en) 1991-12-24 1994-10-11 The President And Fellows Of Harvard College Site-directed mutagenesis of DNA
US5413924A (en) 1992-02-13 1995-05-09 Kosak; Kenneth M. Preparation of wax beads containing a reagent for release by heating
US5241159A (en) 1992-03-11 1993-08-31 Eastman Kodak Company Multi-zone heating for a fuser roller
US6107059A (en) 1992-04-29 2000-08-22 Affymax Technologies N.V. Peptide library and screening method
AU677781B2 (en) 1992-05-01 1997-05-08 Trustees Of The University Of Pennsylvania, The Microfabricated sperm handling devices
AU4222393A (en) 1992-05-01 1993-11-29 Trustees Of The University Of Pennsylvania, The Microfabricated detection structures
AU4222593A (en) 1992-05-01 1993-11-29 Trustees Of The University Of Pennsylvania, The Analysis based on flow restriction
AU4222693A (en) 1992-05-01 1993-11-29 Trustees Of The University Of Pennsylvania, The Microfabricated sperm handling devices
US5427946A (en) 1992-05-01 1995-06-27 Trustees Of The University Of Pennsylvania Mesoscale sperm handling devices
US5726026A (en) 1992-05-01 1998-03-10 Trustees Of The University Of Pennsylvania Mesoscale sample preparation device and systems for determination and processing of analytes
US5587128A (en) 1992-05-01 1996-12-24 The Trustees Of The University Of Pennsylvania Mesoscale polynucleotide amplification devices
WO1993022421A1 (en) 1992-05-01 1993-11-11 Trustees Of The University Of Pennsylvania Microfabricated sperm handling devices
AU4223593A (en) 1992-05-01 1993-11-29 Trustees Of The University Of Pennsylvania, The Polynucleotide amplification analysis using a microfabricated device
AU4222793A (en) 1992-05-01 1993-11-29 Trustees Of The University Of Pennsylvania, The Fluid handling in microfabricated analytical devices
WO1993022053A1 (en) 1992-05-01 1993-11-11 Trustees Of The University Of Pennsylvania Microfabricated detection structures
US5486335A (en) 1992-05-01 1996-01-23 Trustees Of The University Of Pennsylvania Analysis based on flow restriction
US5498392A (en) 1992-05-01 1996-03-12 Trustees Of The University Of Pennsylvania Mesoscale polynucleotide amplification device and method
EP0637996A1 (en) 1992-05-01 1995-02-15 Univ Pennsylvania Microfabricated detection structures.
US5744366A (en) 1992-05-01 1998-04-28 Trustees Of The University Of Pennsylvania Mesoscale devices and methods for analysis of motile cells
AU677197B2 (en) 1992-05-01 1997-04-17 Trustees Of The University Of Pennsylvania, The Polynucleotide amplification analysis using a microfabricated device
WO1993022054A1 (en) 1992-05-01 1993-11-11 Trustees Of The University Of Pennsylvania Analysis based on flow restriction
EP0637997A1 (en) 1992-05-01 1995-02-15 The Trustees Of The University Of Pennsylvania Analysis based on flow restriction
US5296375A (en) 1992-05-01 1994-03-22 Trustees Of The University Of Pennsylvania Mesoscale sperm handling devices
US5635358A (en) 1992-05-01 1997-06-03 Trustees Of The University Of Pennsylvania Fluid handling methods for use in mesoscale analytical devices
AU680195B2 (en) 1992-05-01 1997-07-24 Trustees Of The University Of Pennsylvania, The Analysis based on flow restriction
US5304487A (en) 1992-05-01 1994-04-19 Trustees Of The University Of Pennsylvania Fluid handling in mesoscale analytical devices
WO1993022055A2 (en) 1992-05-01 1993-11-11 Trustees Of The University Of Pennsylvania Fluid handling in microfabricated analytical devices
WO1993022058A1 (en) 1992-05-01 1993-11-11 Trustees Of The University Of Pennsylvania Polynucleotide amplification analysis using a microfabricated device
US5397605A (en) 1992-05-29 1995-03-14 Barbieri; Girolamo Method and apparatus for electrostatically coating a workpiece with paint
US5452955A (en) 1992-06-25 1995-09-26 Vattenfall Utvecking Ab Device for mixing two fluids having different temperatures
US5503851A (en) 1992-07-10 1996-04-02 Ferring Arzneimittel Gmbh Microencapsulation of water-soluble medicaments
JPH0665609A (en) 1992-08-25 1994-03-08 Mitsubishi Materials Corp Manufacturing method of iron-based sintered forged body
US5616478A (en) 1992-10-14 1997-04-01 Chetverin; Alexander B. Method for amplification of nucleic acids in solid media
US6105877A (en) 1992-12-01 2000-08-22 Electrosols Ltd. Dispensing device
US6318640B1 (en) 1992-12-01 2001-11-20 Electrosols, Ltd. Dispensing device
US6105571A (en) 1992-12-22 2000-08-22 Electrosols, Ltd. Dispensing device
WO1994016332A1 (en) 1993-01-13 1994-07-21 Yeda Research And Development Co. Ltd. Method for screening catalytic non-enzyme polypeptides and proteins
WO1994026766A1 (en) 1993-02-19 1994-11-24 Barnes Wayne M Dna polymerases with enhanced thermostability and enhanced length and efficiency of primer extension
JPH06265447A (en) 1993-03-16 1994-09-22 Hitachi Ltd Microreactor and trace component measuring device using the same
US5480614A (en) 1993-03-16 1996-01-02 Hitachi, Ltd. Micro-reactor device for minute sample analysis
DE4308839C2 (en) 1993-03-19 1997-04-30 Jordanow & Co Gmbh Device for mixing flow media
EP0618001A1 (en) 1993-03-31 1994-10-05 Rhone-Poulenc Nutrition Animale Process for preparing active agents containing spherules
JPH07489A (en) 1993-03-31 1995-01-06 Rhone Poulenc Nutrition Animal Preparation of pellet of active component
US5500415A (en) 1993-03-31 1996-03-19 Rhone-Poulenc Nutrition Animale Process for the preparation of spherules of active principles and emulsions containing the spherules
FR2703263A1 (en) 1993-03-31 1994-10-07 Rhone Poulenc Nutrition Animal Process for the preparation of spherules of active principles.
EP0620432A1 (en) 1993-04-15 1994-10-19 Ciba-Geigy Ag Method for controlling sample introduction in microcolumn separation techniques and sampling device
WO1994024314A1 (en) 1993-04-19 1994-10-27 Kauffman Stuart A Random chemistry for the generation of new compounds
WO1994023738A1 (en) 1993-04-19 1994-10-27 Medisorb Technologies International L.P. Encapsulation of nucleic acids with conjugates that facilitate and target cellular uptake and gene expression
US5942056A (en) 1993-04-22 1999-08-24 Federalloy, Inc. Plumbing fixtures and fittings employing copper-bismuth casting alloys
US6268152B1 (en) 1993-06-25 2001-07-31 Affymetrix, Inc. Probe kit for identifying a base in a nucleic acid
US20040091923A1 (en) 1993-07-23 2004-05-13 Bio-Rad Laboratories, Inc. Linked linear amplification of nucleic acids
US5417235A (en) 1993-07-28 1995-05-23 Regents Of The University Of Michigan Integrated microvalve structures with monolithic microflow controller
US5403617A (en) 1993-09-15 1995-04-04 Mobium Enterprises Corporation Hybrid pulsed valve for thin film coating and method
US5512131A (en) 1993-10-04 1996-04-30 President And Fellows Of Harvard College Formation of microstamped patterns on surfaces and derivative articles
US20040159633A1 (en) 1993-10-04 2004-08-19 President & Fellows Of Harvard University Methods of etching articles via micro contact printing
WO1995011922A1 (en) 1993-10-29 1995-05-04 Affymax Technologies N.V. In vitro peptide and antibody display libraries
US6165778A (en) 1993-11-02 2000-12-26 Affymax Technologies N.V. Reaction vessel agitation apparatus
US6316208B1 (en) 1994-01-07 2001-11-13 Memorial Sloan-Kettering Cancer Center Methods for determining isolated p27 protein levels and uses thereof
WO1995019922A1 (en) 1994-01-25 1995-07-27 Ingenieurbüro Von Borries Strip packing
US5643729A (en) 1994-02-24 1997-07-01 Boehringer Ingelheim International Gmbh Methods for diagnosing cancer, precancerous state, or susceptibility to other forms of diseases by detecting an acceleration of exon skipping in IRF-1 mRNA
WO1995024929A2 (en) 1994-03-15 1995-09-21 Brown University Research Foundation Polymeric gene delivery system
US20010020011A1 (en) 1994-03-15 2001-09-06 Edith Mathiowitz Polymeric gene delivery system
US5989815A (en) 1994-03-18 1999-11-23 University Of Utah Research Foundation Methods for detecting predisposition to cancer at the MTS gene
US6068199A (en) 1994-03-29 2000-05-30 Electrosols, Ltd. Dispensing device
US5655517A (en) 1994-03-29 1997-08-12 Electrosols, Ltd. Dispensing device
US5610016A (en) 1994-03-30 1997-03-11 Mochida Pharmaceutical Co., Ltd. Method for measuring adenyl group-containing substances using heteropoly-acid
US5656155A (en) 1994-04-26 1997-08-12 Ip Holding Company Thermophilic aerobic waste treatment system
US5904933A (en) 1994-06-09 1999-05-18 Alliance Pharmaceutical Corp. Stable reverse and multiple fluorocarbon emulsions
WO1995033447A1 (en) 1994-06-09 1995-12-14 Alliance Pharmaceutical Corp. Stable reverse and multiple fluorocarbon emulsions
US5888746A (en) 1994-06-10 1999-03-30 Institute Of Molecular And Cell Biology Tumor diagnosis and prognosis
US5617997A (en) 1994-06-13 1997-04-08 Praxair Technology, Inc. Narrow spray angle liquid fuel atomizers for combustion
US5750988A (en) 1994-07-11 1998-05-12 Hewlett-Packard Company Orthogonal ion sampling for APCI mass spectrometry
US6653626B2 (en) 1994-07-11 2003-11-25 Agilent Technologies, Inc. Ion sampling for APPI mass spectrometry
US5641658A (en) 1994-08-03 1997-06-24 Mosaic Technologies, Inc. Method for performing amplification of nucleic acid with two primers bound to a single solid support
US6124439A (en) 1994-08-17 2000-09-26 The Rockefeller University OB polypeptide antibodies and method of making
US5935331A (en) 1994-09-09 1999-08-10 Matsushita Electric Industrial Co., Ltd. Apparatus and method for forming films
US5762775A (en) 1994-09-21 1998-06-09 Lockheed Martin Energy Systems, Inc. Method for electrically producing dispersions of a nonconductive fluid in a conductive medium
US5995341A (en) 1994-09-30 1999-11-30 Kabushiki Kaisha Toshiba Magnetic disk drive recording a signal with a skew angle
US5695934A (en) 1994-10-13 1997-12-09 Lynx Therapeutics, Inc. Massively parallel sequencing of sorted polynucleotides
US5846719A (en) 1994-10-13 1998-12-08 Lynx Therapeutics, Inc. Oligonucleotide tags for sorting and identification
US6172214B1 (en) 1994-10-13 2001-01-09 Lynx Therapeutics, Inc. Oligonucleotide tags for sorting and identification
US6138077A (en) 1994-10-13 2000-10-24 Lynx Therapeutics, Inc. Method, apparatus and computer program product for determining a set of non-hybridizing oligonucleotides
US6172218B1 (en) 1994-10-13 2001-01-09 Lynx Therapeutics, Inc. Oligonucleotide tags for sorting and identification
US6352828B1 (en) 1994-10-13 2002-03-05 Lynx Therapeutics, Inc. Oligonucleotide tags for sorting and identification
US5863722A (en) 1994-10-13 1999-01-26 Lynx Therapeutics, Inc. Method of sorting polynucleotides
US6235475B1 (en) 1994-10-13 2001-05-22 Lynx Therapeutics, Inc. Oligonucleotide tags for sorting and identification
US6150516A (en) 1994-10-13 2000-11-21 Lynx Therapeutics, Inc. Kits for sorting and identifying polynucleotides
US5604097A (en) 1994-10-13 1997-02-18 Spectragen, Inc. Methods for sorting polynucleotides using oligonucleotide tags
JPH08153669A (en) 1994-11-30 1996-06-11 Hitachi Ltd Thin film forming method and forming apparatus
US5813988A (en) 1995-02-03 1998-09-29 Research Foundation Time-resolved diffusion tomographic imaging in highly scattering turbid media
US5661222A (en) 1995-04-13 1997-08-26 Dentsply Research & Development Corp. Polyvinylsiloxane impression material
WO1996034112A1 (en) 1995-04-24 1996-10-31 Chromaxome Corp. Methods for generating and screening novel metabolic pathways
WO1996038730A1 (en) 1995-06-02 1996-12-05 Cdc Technologies, Inc. Apparatus and method for mixing fluids for analysis
US5887755A (en) 1995-06-06 1999-03-30 Quantic Biomedical Partners Wound sealant preparation and application device and method
WO1996040062A1 (en) 1995-06-07 1996-12-19 Georgetown University A method of transfection of cells using liposomally encapsulated nucleic acids
WO1996040057A2 (en) 1995-06-07 1996-12-19 Alliance Pharmaceutical Corp. Reverse fluorocarbon emulsion compositions for drug delivery
WO1996040723A1 (en) 1995-06-07 1996-12-19 The General Hospital Corporation Catalytic dna
US6207372B1 (en) 1995-06-07 2001-03-27 Genzyme Corporation Universal primer sequence for multiplex DNA amplification
US5882856A (en) 1995-06-07 1999-03-16 Genzyme Corporation Universal primer sequence for multiplex DNA amplification
US5989892A (en) 1995-06-14 1999-11-23 Tonen Corporation Microorganisms, demulsifiers and processes for breaking an emulsion
US5932100A (en) 1995-06-16 1999-08-03 University Of Washington Microfabricated differential extraction device and method
WO1997000442A1 (en) 1995-06-16 1997-01-03 The University Of Washington Microfabricated differential extraction device and method
WO1997000125A1 (en) 1995-06-16 1997-01-03 Novartis Ag Flow cell for the passive mixing of flowable substances
US5589136A (en) 1995-06-20 1996-12-31 Regents Of The University Of California Silicon-based sleeve devices for chemical reactions
US20020022261A1 (en) 1995-06-29 2002-02-21 Anderson Rolfe C. Miniaturized genetic analysis systems and methods
US5789206A (en) 1995-07-07 1998-08-04 Myriad Genetics, Inc. Method for ligating adaptors to nucleic acids which methods are useful for obtaining the ends of genes
US6124388A (en) 1995-07-19 2000-09-26 Nippon Telegraph And Telephone Corporation Water repellent composition, fluorocarbon polymer coating composition and coating film therefrom
US5872010A (en) 1995-07-21 1999-02-16 Northeastern University Microscale fluid handling system
WO1997004297A1 (en) 1995-07-21 1997-02-06 Northeastern University Microscale fluid handling system
WO1997004748A2 (en) 1995-08-01 1997-02-13 Advanced Therapies, Inc. Enhanced artificial viral envelopes for cellular delivery of therapeutic substances
US5636400A (en) 1995-08-07 1997-06-10 Young; Keenan L. Automatic infant bottle cleaner
US6130098A (en) 1995-09-15 2000-10-10 The Regents Of The University Of Michigan Moving microdroplets
US5849491A (en) 1995-09-22 1998-12-15 Terragen Diversity Inc. Method for isolating xylanase gene sequences from soil DNA, compositions useful in such method and compositions obtained thereby
US5851769A (en) 1995-09-27 1998-12-22 The Regents Of The University Of California Quantitative DNA fiber mapping
US6243373B1 (en) 1995-11-01 2001-06-05 Telecom Internet Ltd. Method and apparatus for implementing a computer network/internet telephone system
US6638749B1 (en) 1995-11-13 2003-10-28 Genencor International, Inc. Carbon dioxide soluble surfactant having two fluoroether CO2-philic tail groups and a head group
US5882680A (en) 1995-12-07 1999-03-16 Freund Industrial Co., Ltd. Seamless capsule and method of manufacturing the same
US6310653B1 (en) 1995-12-12 2001-10-30 Ronald D. Malcolm, Jr. Phase comparison and phase adjustment for synchronization to a reference signal that is asynchronous with respect to a digital sampling clock
US5733526A (en) 1995-12-14 1998-03-31 Alliance Pharmaceutical Corp. Hydrocarbon oil/fluorochemical preparations and methods of use
WO1997023140A1 (en) 1995-12-18 1997-07-03 Abbott Laboratories Stabilization of liquid nutritional products comprising soy polysaccharide
US5681600A (en) 1995-12-18 1997-10-28 Abbott Laboratories Stabilization of liquid nutritional products and method of making
US6261797B1 (en) 1996-01-29 2001-07-17 Stratagene Primer-mediated polynucleotide synthesis and manipulation techniques
WO1997028556A1 (en) 1996-01-31 1997-08-07 Hewlett-Packard Company Apparatus for forming liquid droplets having a mechanically fixed inner microtube
US5868322A (en) 1996-01-31 1999-02-09 Hewlett-Packard Company Apparatus for forming liquid droplets having a mechanically fixed inner microtube
JP2001517353A (en) 1996-01-31 2001-10-02 アジレント・テクノロジーズ・インク Droplet generator with mechanically fixed internal microtubules
US6155710A (en) 1996-02-20 2000-12-05 Japan, As Represented By Director Of National Food Research Institute, Ministry Of Agriculture, Forestry And Fisheries Method and device for producing emulsions
US8012382B2 (en) 1996-03-15 2011-09-06 President And Fellows Of Harvard College Molded waveguides
US6355198B1 (en) 1996-03-15 2002-03-12 President And Fellows Of Harvard College Method of forming articles including waveguides via capillary micromolding and microtransfer molding
WO1997038318A1 (en) 1996-04-04 1997-10-16 Novartis Ag Device for counting small particles and a sorting apparatus comprising such a device
US5739036A (en) 1996-04-15 1998-04-14 Dade International Inc. Method for analysis
US6207397B1 (en) 1996-04-18 2001-03-27 Ariad Pharmaceuticals, Inc. In vitro fluorescence polarization assay
WO1997039814A1 (en) 1996-04-19 1997-10-30 Central Research Laboratories Limited Method and apparatus for diffusive transfer between immiscible liquids
US5783431A (en) 1996-04-24 1998-07-21 Chromaxome Corporation Methods for generating and screening novel metabolic pathways
WO1997040141A2 (en) 1996-04-25 1997-10-30 Medical Research Council Isolation of enzymes
US6184012B1 (en) 1996-04-25 2001-02-06 Medical Research Council Isolation of enzymes
US6357670B2 (en) 1996-05-13 2002-03-19 Universidad De Sevilla Stabilized capillary microjet and devices and methods for producing same
US6554202B2 (en) 1996-05-13 2003-04-29 Universidad De Sevilla Fuel injection nozzle and method of use
US6241159B1 (en) 1996-05-13 2001-06-05 Universidad De Sevilla Liquid atomization procedure
US6234402B1 (en) 1996-05-13 2001-05-22 Universidad De Sevilla Stabilized capillary microjet and devices and methods for producing same
US6464886B2 (en) 1996-05-13 2002-10-15 Universidad De Sevilla Device and method for creating spherical particles of uniform size
US20020033422A1 (en) 1996-05-13 2002-03-21 Ganan-Calvo Alfonso M. Fuel injection nozzle and method of use
US6386463B1 (en) 1996-05-13 2002-05-14 Universidad De Sevilla Fuel injection nozzle and method of use
US20010010338A1 (en) 1996-05-13 2001-08-02 Alfonso Ganan-Calvo Device and method for creating spherical particles of uniform size
US20010042793A1 (en) 1996-05-13 2001-11-22 Alfonso Ganan-Calvo Stabilized capillary microjet and devices and methods for producing same
US6557834B2 (en) 1996-05-13 2003-05-06 Universidad De Seville Device and method for fluid aeration via gas forced through a liquid within an orifice of a pressure chamber
US6197835B1 (en) 1996-05-13 2001-03-06 Universidad De Sevilla Device and method for creating spherical particles of uniform size
US6432148B1 (en) 1996-05-13 2002-08-13 Universidad De Sevilla Fuel injection nozzle and method of use
US6196525B1 (en) 1996-05-13 2001-03-06 Universidad De Sevilla Device and method for fluid aeration via gas forced through a liquid within an orifice of a pressure chamber
US6174469B1 (en) 1996-05-13 2001-01-16 Universidad De Sevilla Device and method for creating dry particles
US6299145B1 (en) 1996-05-13 2001-10-09 Universidad De Sevilla Device and method for fluid aeration via gas forced through a liquid within an orifice of a pressure chamber
US6119953A (en) 1996-05-13 2000-09-19 Aradigm Corporation Liquid atomization process
US6116516A (en) 1996-05-13 2000-09-12 Universidad De Sevilla Stabilized capillary microjet and devices and methods for producing same
US6394429B2 (en) 1996-05-13 2002-05-28 Universidad De Sevilla Device and method for fluid aeration via gas forced through a liquid within an orifice of a pressure chamber
US6189803B1 (en) 1996-05-13 2001-02-20 University Of Seville Fuel injection nozzle and method of use
US6187214B1 (en) 1996-05-13 2001-02-13 Universidad De Seville Method and device for production of components for microfabrication
US6405936B1 (en) 1996-05-13 2002-06-18 Universidad De Sevilla Stabilized capillary microjet and devices and methods for producing same
US20070042419A1 (en) 1996-05-29 2007-02-22 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
AU2935197A (en) 1996-05-31 1998-01-05 University Of Washington Valveless liquid microswitch
US5726404A (en) 1996-05-31 1998-03-10 University Of Washington Valveless liquid microswitch
WO1997045644A1 (en) 1996-05-31 1997-12-04 The University Of Washington Valveless liquid microswitch
US5840506A (en) 1996-06-05 1998-11-24 Thomas Jefferson University Methods for the diagnosis and prognosis of cancer
WO1997047763A1 (en) 1996-06-14 1997-12-18 Curagen Corporation Identification and comparison of protein-protein interactions and inhibitors thereof
US5876771A (en) 1996-06-20 1999-03-02 Tetra Laval Holdings & Finance, Sa Process and article for determining the residence time of a food particle
US6150180A (en) 1996-06-28 2000-11-21 Caliper Technologies Corp. High throughput screening assay systems in microscale fluidic devices
US5779868A (en) 1996-06-28 1998-07-14 Caliper Technologies Corporation Electropipettor and compensation means for electrophoretic bias
WO1998000231A1 (en) 1996-06-28 1998-01-08 Caliper Technologies Corporation High-throughput screening assay systems in microscale fluidic devices
CA2258481A1 (en) 1996-06-28 1998-01-08 Caliper Technologies Corporation Electropipettor and compensation means for electrophoretic bias
US6046056A (en) 1996-06-28 2000-04-04 Caliper Technologies Corporation High throughput screening assay systems in microscale fluidic devices
US6399389B1 (en) 1996-06-28 2002-06-04 Caliper Technologies Corp. High throughput screening assay systems in microscale fluidic devices
US6042709A (en) 1996-06-28 2000-03-28 Caliper Technologies Corp. Microfluidic sampling system and methods
WO1998000705A1 (en) 1996-06-28 1998-01-08 Caliper Technologies Corporation Electropipettor and compensation means for electrophoretic bias
US6274337B1 (en) 1996-06-28 2001-08-14 Caliper Technologies Corp. High throughput screening assay systems in microscale fluidic devices
US6429025B1 (en) 1996-06-28 2002-08-06 Caliper Technologies Corp. High-throughput screening assay systems in microscale fluidic devices
AU3499097A (en) 1996-06-28 1998-01-21 Caliper Technologies Corporation High throughput screening assay systems in microscale fluidic devices
US5880071A (en) 1996-06-28 1999-03-09 Caliper Technologies Corporation Electropipettor and compensation means for electrophoretic bias
US6080295A (en) 1996-06-28 2000-06-27 Caliper Technologies Corporation Electropipettor and compensation means for electrophoretic bias
US5942443A (en) 1996-06-28 1999-08-24 Caliper Technologies Corporation High throughput screening assay systems in microscale fluidic devices
BR9710052A (en) 1996-06-28 2000-01-11 Caliper Techn Corp Microfluid system with compensation for electrophoretic polarization, electropipettor, processes for introducing materials from a series of sources into a microfluidic system, for controllably distributing a fluid stream and for transporting fluid samples, sampling system, use of a substrate , use of a microfluidic system, and substrate.
US6267858B1 (en) 1996-06-28 2001-07-31 Caliper Technologies Corp. High throughput screening assay systems in microscale fluidic devices
US5958203A (en) 1996-06-28 1999-09-28 Caliper Technologies Corportion Electropipettor and compensation means for electrophoretic bias
US5972187A (en) 1996-06-28 1999-10-26 Caliper Technologies Corporation Electropipettor and compensation means for electrophoretic bias
US6630353B1 (en) 1996-06-28 2003-10-07 Caliper Technologies Corp. High throughput screening assay systems in microscale fluidic devices
US6306659B1 (en) 1996-06-28 2001-10-23 Caliper Technologies Corp. High throughput screening assay systems in microscale fluidic devices
US6558944B1 (en) 1996-06-28 2003-05-06 Caliper Technologies Corp. High throughput screening assay systems in microscale fluidic devices
US6558960B1 (en) 1996-06-28 2003-05-06 Caliper Technologies Corp. High throughput screening assay systems in microscale fluidic devices
WO1998002237A1 (en) 1996-07-15 1998-01-22 Kemgas Limited Production of powders
US6252129B1 (en) 1996-07-23 2001-06-26 Electrosols, Ltd. Dispensing device and method for forming material
US5670325A (en) 1996-08-14 1997-09-23 Exact Laboratories, Inc. Method for the detection of clonal populations of transformed cells in a genomically heterogeneous cellular sample
US6203993B1 (en) 1996-08-14 2001-03-20 Exact Science Corp. Methods for the detection of nucleic acids
US6146828A (en) 1996-08-14 2000-11-14 Exact Laboratories, Inc. Methods for detecting differences in RNA expression levels and uses therefor
US6214558B1 (en) 1996-08-14 2001-04-10 Exact Laboratories, Inc. Methods for the detection of chromosomal aberrations
WO1998010267A1 (en) 1996-09-04 1998-03-12 Technical University Of Denmark A micro flow system for particle separation and analysis
US6432630B1 (en) 1996-09-04 2002-08-13 Scandinanian Micro Biodevices A/S Micro-flow system for particle separation and analysis
US5884846A (en) 1996-09-19 1999-03-23 Tan; Hsiaoming Sherman Pneumatic concentric nebulizer with adjustable and capillaries
US6221654B1 (en) 1996-09-25 2001-04-24 California Institute Of Technology Method and apparatus for analysis and sorting of polynucleotides based on size
US6344325B1 (en) 1996-09-25 2002-02-05 California Institute Of Technology Methods for analysis and sorting of polynucleotides
US5858187A (en) 1996-09-26 1999-01-12 Lockheed Martin Energy Systems, Inc. Apparatus and method for performing electrodynamic focusing on a microchip
US6120666A (en) 1996-09-26 2000-09-19 Ut-Battelle, Llc Microfabricated device and method for multiplexed electrokinetic focusing of fluid streams and a transport cytometry method using same
WO1998013502A2 (en) 1996-09-27 1998-04-02 Icos Corporation Method to identify compounds for disrupting protein/protein interactions
US6210891B1 (en) 1996-09-27 2001-04-03 Pyrosequencing Ab Method of sequencing DNA
US6140053A (en) 1996-11-06 2000-10-31 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US20050251049A1 (en) 1996-11-19 2005-11-10 Astron Clinica Limited Method of and apparatus for investigating tissue histology
US6324417B1 (en) 1996-11-19 2001-11-27 Optiscan Limited Method for measurement of skin histology
WO1998022625A1 (en) 1996-11-20 1998-05-28 The Regents Of The University Of Michigan Microfabricated isothermal nucleic acid amplification devices and methods
WO1998023733A2 (en) 1996-11-27 1998-06-04 University Of Washington Thermostable polymerases having altered fidelity
US6310354B1 (en) 1996-12-03 2001-10-30 Erkki Soini Method and a device for monitoring nucleic acid amplification reactions
US6309842B1 (en) 1996-12-03 2001-10-30 Glaxo Wellcome Inc. Use of modified tethers in screening compound libraries
US6258568B1 (en) 1996-12-23 2001-07-10 Pyrosequencing Ab Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation
US20030104372A1 (en) 1996-12-23 2003-06-05 Pyrosequencing Ab. Allele specific primer extension
WO1998031700A1 (en) 1997-01-21 1998-07-23 The General Hospital Corporation Selection of proteins using rna-protein fusions
US6235383B1 (en) 1997-01-24 2001-05-22 Samsung Corning Co., Ltd. Glass article having a durable water repellent surface
WO1998033001A1 (en) 1997-01-29 1998-07-30 The Board Of Trustees Of The Leland Stanford Junior University Micromachined fluidic coupler
WO1998034120A1 (en) 1997-01-31 1998-08-06 Universite De Montreal Protein fragment complementation assays to detect biomolecular interactions
US5921678A (en) 1997-02-05 1999-07-13 California Institute Of Technology Microfluidic sub-millisecond mixers
JPH10217477A (en) 1997-02-07 1998-08-18 Fuji Xerox Co Ltd Ink jet recording device
US20020119455A1 (en) 1997-02-12 2002-08-29 Chan Eugene Y. Methods and products for analyzing polymers
WO1998037186A1 (en) 1997-02-18 1998-08-27 Actinova Limited In vitro peptide or protein expression library
US6307957B1 (en) 1997-02-28 2001-10-23 Electro-Optical Sciences Inc Multispectral imaging and characterization of biological tissue
US6208749B1 (en) 1997-02-28 2001-03-27 Electro-Optical Sciences, Inc. Systems and methods for the multispectral imaging and characterization of skin tissue
US6081612A (en) 1997-02-28 2000-06-27 Electro Optical Sciences Inc. Systems and methods for the multispectral imaging and characterization of skin tissue
US20020034737A1 (en) 1997-03-04 2002-03-21 Hyseq, Inc. Methods and compositions for detection or quantification of nucleic acid species
US6045755A (en) 1997-03-10 2000-04-04 Trega Biosciences,, Inc. Apparatus and method for combinatorial chemistry synthesis
US6280948B1 (en) 1997-03-11 2001-08-28 Wisconsin Alumni Research Foundation Nucleic acid indexing
US6023540A (en) 1997-03-14 2000-02-08 Trustees Of Tufts College Fiber optic sensor with encoded microspheres
US6266459B1 (en) 1997-03-14 2001-07-24 Trustees Of Tufts College Fiber optic sensor with encoded microspheres
WO1998041869A1 (en) 1997-03-18 1998-09-24 Chromaxome Corporation Methods for screening compounds using encapsulated cells
US6316213B1 (en) 1997-03-19 2001-11-13 The Board Of Trustees Of The University Of Arkansas Methods for the early diagnosis of ovarian, breast and lung cancer
US6294344B1 (en) 1997-03-19 2001-09-25 The Board Of Trustees Of The University Of Arkansas Methods for the early diagnosis of ovarian cancer
US6268165B1 (en) 1997-03-19 2001-07-31 The Board Of Trustees Of The University Of Arkansas Methods for the early diagnosis of ovarian cancer
US6048551A (en) 1997-03-27 2000-04-11 Hilfinger; John M. Microsphere encapsulation of gene transfer vectors
US6553960B1 (en) 1997-04-11 2003-04-29 Yanmar Co., Ltd. Combustion system for direct injection diesel engines
US6143496A (en) 1997-04-17 2000-11-07 Cytonix Corporation Method of sampling, amplifying and quantifying segment of nucleic acid, polymerase chain reaction assembly having nanoliter-sized sample chambers, and method of filling assembly
US8067159B2 (en) 1997-04-17 2011-11-29 Applied Biosystems, Llc Methods of detecting amplified product
US6391559B1 (en) 1997-04-17 2002-05-21 Cytonix Corporation Method of sampling, amplifying and quantifying segment of nucleic acid, polymerase chain reaction assembly having nanoliter-sized sample chambers, and method of filling assembly
US8278071B2 (en) 1997-04-17 2012-10-02 Applied Biosystems, Llc Method for detecting the presence of a single target nucleic acid in a sample
US8257925B2 (en) 1997-04-17 2012-09-04 Applied Biosystems, Llc Method for detecting the presence of a single target nucleic acid in a sample
US6180372B1 (en) 1997-04-23 2001-01-30 Bruker Daltonik Gmbh Method and devices for extremely fast DNA replication by polymerase chain reactions (PCR)
US6130052A (en) 1997-04-25 2000-10-10 Ludwig Institute For Cancer Research Leukemia associated genes
US6028066A (en) 1997-05-06 2000-02-22 Imarx Pharmaceutical Corp. Prodrugs comprising fluorinated amphiphiles
US6251661B1 (en) 1997-05-14 2001-06-26 Morishita Jintan Co., Ltd. Seamless capsule for synthesizing biopolymer and method for producing the same
WO1998052691A1 (en) 1997-05-16 1998-11-26 Alberta Research Council Microfluidic system and methods of use
US6432143B2 (en) 1997-05-16 2002-08-13 Life Technologies, Inc. Automated liquid manufacturing system
US6632619B1 (en) 1997-05-16 2003-10-14 The Governors Of The University Of Alberta Microfluidic system and methods of use
US6475441B1 (en) 1997-06-09 2002-11-05 Caliper Technologies Corp. Method for in situ concentration and/or dilution of materials in microfluidic systems
US5888778A (en) 1997-06-16 1999-03-30 Exact Laboratories, Inc. High-throughput screening method for identification of genetic mutations or disease-causing microorganisms using segmented primers
US20010034031A1 (en) 1997-06-16 2001-10-25 Recombinant Biocatalysis Inc. Delaware Corporation High throughput screening for novel enzymes
US5928870A (en) 1997-06-16 1999-07-27 Exact Laboratories, Inc. Methods for the detection of loss of heterozygosity
US20020015997A1 (en) 1997-06-16 2002-02-07 Lafferty William Michael Capillary array-based sample screening
WO1998058085A1 (en) 1997-06-16 1998-12-23 Diversa Corporation High throughput screening for novel enzymes
US6074879A (en) 1997-06-23 2000-06-13 Bayer Corporation Synthetic polymer particles for use as standards and calibrators in flow cytometry
US6118849A (en) 1997-06-27 2000-09-12 Japan Science And Technology Corporation Microstrip gas chamber high-speed data acquisition system and method of measuring samples by use of the system
US20070259374A1 (en) 1997-07-07 2007-11-08 Medical Research Council In vitro sorting method
WO1999002671A1 (en) 1997-07-07 1999-01-21 Medical Research Council In vitro sorting method
US20050042648A1 (en) 1997-07-07 2005-02-24 Andrew Griffiths Vitro sorting method
US20070077579A1 (en) 1997-07-07 2007-04-05 Andrew Griffiths In vitro sorting method
US7138233B2 (en) 1997-07-07 2006-11-21 Medical Research Council IN vitro sorting method
US8367326B2 (en) 1997-07-07 2013-02-05 Medical Research Council In vitro sorting method
US7252943B2 (en) 1997-07-07 2007-08-07 Medical Research Council In Vitro sorting method
US20030124586A1 (en) 1997-07-07 2003-07-03 Andrew Griffiths In vitro sorting method
US7582446B2 (en) 1997-07-07 2009-09-01 Medical Research Council In vitro sorting method
US6489103B1 (en) 1997-07-07 2002-12-03 Medical Research Council In vitro sorting method
US9528106B2 (en) 1997-07-07 2016-12-27 Medical Research Council In vitro sorting method
US7638276B2 (en) 1997-07-07 2009-12-29 454 Life Sciences Corporation In vitro sorting method
US20050069920A1 (en) 1997-07-07 2005-03-31 Andrew Griffiths In vitro sorting method
EP0895120A1 (en) 1997-07-15 1999-02-03 Konica Corporation Method for preparing silver halide emulsion
US6096495A (en) 1997-07-15 2000-08-01 Konica Corporation Method for preparing silver halide emulsion
US6261661B1 (en) 1997-07-23 2001-07-17 Matsushita Electrical Industrial Co., Ltd. Optical information recording medium and method for producing the same
US5980936A (en) 1997-08-07 1999-11-09 Alliance Pharmaceutical Corp. Multiple emulsions comprising a hydrophobic continuous phase
US6627603B1 (en) 1997-08-07 2003-09-30 Centre National De La Recherche Scientifiquue (C.N.R.S.) Method for releasing an active principle contained a multiple emulsion
US6197335B1 (en) 1997-09-16 2001-03-06 Bernard Charles Sherman Solid pharmaceutical compositions comprising a cyclosporin and an anionic surfactant
US20020005354A1 (en) 1997-09-23 2002-01-17 California Institute Of Technology Microfabricated cell sorter
US20080176211A1 (en) 1997-09-23 2008-07-24 California Institute Of Technology Microfabricated Cell Sorter
US6833242B2 (en) 1997-09-23 2004-12-21 California Institute Of Technology Methods for detecting and sorting polynucleotides based on size
US6540895B1 (en) 1997-09-23 2003-04-01 California Institute Of Technology Microfabricated cell sorter for chemical and biological materials
US6103537A (en) 1997-10-02 2000-08-15 Aclara Biosciences, Inc. Capillary assays involving separation of free and bound species
US6403373B1 (en) 1997-10-10 2002-06-11 Ludwig Institute For Cancer Research Isolated nucleic acid molecules associated with colon, renal, and stomach cancer and methods of using these
US6511803B1 (en) 1997-10-10 2003-01-28 President And Fellows Of Harvard College Replica amplification of nucleic acid arrays
US6008003A (en) 1997-10-28 1999-12-28 Promega Corporation Non-invasive diagnostic method for interstitial cystitis and bladder cancer
WO1999022858A1 (en) 1997-11-05 1999-05-14 British Nuclear Fuels Plc Reactions of aromatic compounds
US6162421A (en) 1997-11-17 2000-12-19 Revlon Consumer Products Corporation Pigmented water-in-oil emulsion cosmetic sticks
AU1276099A (en) 1997-12-01 1999-06-16 Minnesota Mining And Manufacturing Company Process for production of heat sensitive dispersions or emulsions
WO1999028020A1 (en) 1997-12-01 1999-06-10 Minnesota Mining And Manufacturing Company Process for production of heat sensitive dispersions or emulsions
US20040053247A1 (en) 1997-12-01 2004-03-18 Sloan-Kettering Institute For Cancer Research Markers for prostate cancer
US5927852A (en) 1997-12-01 1999-07-27 Minnesota Mining And Manfacturing Company Process for production of heat sensitive dispersions or emulsions
WO1999028507A1 (en) 1997-12-04 1999-06-10 Exact Laboratories, Inc. Methods for the detection of chromosomal aberrations
WO1999031019A1 (en) 1997-12-17 1999-06-24 Universidad De Sevilla Device and method for creating spherical particles of uniform size
US6171796B1 (en) 1998-01-21 2001-01-09 Urocor, Inc. Biomarkers and targets for diagnosis prognosis and management of prostate disease
US6268222B1 (en) 1998-01-22 2001-07-31 Luminex Corporation Microparticles attached to nanoparticles labeled with flourescent dye
WO1999042539A1 (en) 1998-02-17 1999-08-26 Sofitech N.V. Anti-accretion additives for drilling fluids
US6503933B1 (en) 1998-02-19 2003-01-07 Aventis Cropscience Uk Limited 2-pyridylmethylamine derivatives useful as fungicides
US7291462B2 (en) 1998-02-20 2007-11-06 The University Of Arkansas For Medical Sciences TADG-15: an extracellular serine protease overexpressed in carcinomas
US6137214A (en) 1998-02-23 2000-10-24 Micron Technology, Inc. Display device with silicon-containing adhesion layer
US6897018B1 (en) 1998-02-25 2005-05-24 The United States Of America As Represented By The Department Of Health And Human Services DLC-1 gene deleted in cancers
US6292756B1 (en) 1998-02-26 2001-09-18 Premier Instruments, Inc. Narrow band infrared water fraction apparatus for gas well and liquid hydrocarbon flow stream use
US6679441B1 (en) 1998-03-27 2004-01-20 Centre National De La Recherche Scientifique (C.N.R.S.) Electrohydrodynamic spraying means
US6177479B1 (en) 1998-03-30 2001-01-23 Japan As Represented By Director Of National Food Research Institute, Ministry Of Agriculture, Forestry And Fisheries Continuous manufacturing method for microspheres and apparatus
US6336463B1 (en) 1998-03-31 2002-01-08 Nec Corporation Cleaning/drying station and production line for semiconductor devices
WO1999054730A1 (en) 1998-04-20 1999-10-28 Wallac Oy Method and device for carrying out a chemical analysis in small amounts of liquid
US6814980B2 (en) 1998-04-23 2004-11-09 The Regents Of The University Of Michigan Microspheres containing condensed polyanionic bioactive agents and methods for their production
US20060269558A1 (en) 1998-04-27 2006-11-30 Murphy Gerald P Nr-CAM gene, nucleic acids and nucleic acid products for therapeutic and diagnostic uses for tumors
US5997636A (en) 1998-05-01 1999-12-07 Instrumentation Technology Associates, Inc. Method and apparatus for growing crystals
US6646253B1 (en) 1998-05-20 2003-11-11 GSF-Forschungszentrum für Umwelt und Gesundheit GmbH Gas inlet for an ion source
AU4955799A (en) 1998-05-22 1999-12-13 California Institute Of Technology Microfabricated cell sorter
WO1999061888A2 (en) 1998-05-22 1999-12-02 California Institute Of Technology Microfabricated cell sorter
US6659370B1 (en) 1998-05-25 2003-12-09 Fuji Bc Engineering Co., Ltd. Liquid spray device and cutting method
WO1999063260A1 (en) 1998-06-04 1999-12-09 The Regents Of The University Of California Microfluidic interconnects
US6551836B1 (en) 1998-06-08 2003-04-22 Caliper Technologies Corp. Microfluidic devices, systems and methods for performing integrated reactions and separations
AU747464B2 (en) 1998-06-08 2002-05-16 Caliper Technologies Corporation Microfluidic devices, systems and methods for performing integrated reactions and separations
US6326145B1 (en) 1998-06-13 2001-12-04 Zeneca Limited Methods for detecting target nucleic acid sequences
US6576420B1 (en) 1998-06-23 2003-06-10 Regents Of The University Of California Method for early diagnosis of, and determination of prognosis in, cancer
US20030083276A1 (en) 1998-06-30 2003-05-01 Li Gloria C. Uses of DNA-PK
US6258858B1 (en) 1998-07-02 2001-07-10 Japan As Represented By Director Of National Food Research Institute, Ministry Of Agriculture, Forestry And Fisheries Cross-flow microchannel apparatus and method of producing or separating emulsions making use thereof
WO2000004139A1 (en) 1998-07-17 2000-01-27 Mirus Corporation Micellar systems
US6227466B1 (en) 1998-08-04 2001-05-08 William J. Hartman Electrostatic spray module
US20070020617A1 (en) 1998-08-07 2007-01-25 Cellay, Llc C/O One Cell Systems, Inc. Gel microdrops in genetic analysis
US6210896B1 (en) 1998-08-13 2001-04-03 Us Genomics Molecular motors
US20020158027A1 (en) 1998-09-17 2002-10-31 Moon James E. Integrated monolithic microfabricated electrospray and liquid chromatography system and method
US20010023078A1 (en) 1998-09-18 2001-09-20 Massachusetts Institute Of Technology Biological applications of quantum dots
US6844377B1 (en) 1998-09-30 2005-01-18 Basf Aktiengesellschaft Polymer particles containing dye
US7229770B1 (en) 1998-10-01 2007-06-12 The Regents Of The University Of California YKL-40 as a marker and prognostic indicator for cancers
US6637463B1 (en) 1998-10-13 2003-10-28 Biomicro Systems, Inc. Multi-channel microfluidic system design with balanced fluid flow distribution
US6601613B2 (en) 1998-10-13 2003-08-05 Biomicro Systems, Inc. Fluid circuit components based upon passive fluid dynamics
US6591852B1 (en) 1998-10-13 2003-07-15 Biomicro Systems, Inc. Fluid circuit components based upon passive fluid dynamics
US6296020B1 (en) 1998-10-13 2001-10-02 Biomicro Systems, Inc. Fluid circuit components based upon passive fluid dynamics
US20020036018A1 (en) 1998-10-13 2002-03-28 Mcneely Michael R. Fluid circuit components based upon passive fluid dynamics
US7364862B2 (en) 1998-10-19 2008-04-29 Diadexus, Inc. Method of diagnosing, monitoring, staging, imaging and treating prostate cancer
US7432064B2 (en) 1998-10-19 2008-10-07 Diadexus, Inc. Method of diagnosing, monitoring, staging, imaging and treating prostate cancer
US7022472B2 (en) 1998-10-22 2006-04-04 Diadexus, Inc. Mutations in human MLH1 and human MSH2 genes useful in diagnosing colorectal cancer
US6086740A (en) 1998-10-29 2000-07-11 Caliper Technologies Corp. Multiplexed microfluidic devices and systems
US6569631B1 (en) 1998-11-12 2003-05-27 3-Dimensional Pharmaceuticals, Inc. Microplate thermal shift assay for ligand development using 5-(4″dimethylaminophenyl)-2-(4′-phenyl)oxazole derivative fluorescent dyes
US6614598B1 (en) 1998-11-12 2003-09-02 Institute Of Technology, California Microlensing particles and applications
US6450189B1 (en) 1998-11-13 2002-09-17 Universidad De Sevilla Method and device for production of components for microfabrication
US6139303A (en) 1998-11-20 2000-10-31 United Technologies Corporation Fixture for disposing a laser blocking material in an airfoil
US20020084417A1 (en) 1998-11-23 2002-07-04 Khalil Omar S. Non-invasive sensor capable of determining optical parameters in a sample having multiple layers
US20020142344A1 (en) 1998-12-11 2002-10-03 Mark Akeson Targeted molecular bar codes and methods for using the same
US6399339B1 (en) 1998-12-14 2002-06-04 Forschungszentrum Julich Gmbh Method for the enantioselective reduction of 3,5-dioxocarboxylic acids, their salts and their esters
US6248378B1 (en) 1998-12-16 2001-06-19 Universidad De Sevilla Enhanced food products
WO2000037924A1 (en) 1998-12-22 2000-06-29 Research Foundation Of Cuny Time-resolved optical backscattering tomographic image reconstruction in scattering turbid media
WO2000040712A1 (en) 1999-01-07 2000-07-13 Medical Research Council Optical sorting method
US20050037392A1 (en) 1999-01-07 2005-02-17 Andrew Griffiths Optical sorting method
US20090325236A1 (en) 1999-01-07 2009-12-31 Andrew Griffiths Optical sorting method
US20090053700A1 (en) 1999-01-07 2009-02-26 Andrew Griffiths Optical sorting method
US6808882B2 (en) 1999-01-07 2004-10-26 Medical Research Council Optical sorting method
US7897341B2 (en) 1999-01-07 2011-03-01 Medical Research Council Optical sorting method
US20050164239A1 (en) 1999-01-07 2005-07-28 Andrew Griffiths Optical sorting method
US20020119459A1 (en) 1999-01-07 2002-08-29 Andrew Griffiths Optical sorting method
US20010034025A1 (en) 1999-01-15 2001-10-25 Ljl Biosystems, Inc. Methods and apparatus for detecting polynucleotide hybridization
US20040031688A1 (en) 1999-01-25 2004-02-19 Shenderov Alexander David Actuators for microfluidics without moving parts
US20060003429A1 (en) 1999-01-29 2006-01-05 Frost John W Biocatalytic synthesis of quinic acid and conversion to hydroquinone
WO2000047322A2 (en) 1999-02-12 2000-08-17 Board Of Regents, The University Of Texas System Method and apparatus for programmable fluidic processing
US20020036139A1 (en) 1999-02-12 2002-03-28 Board Of Regents, The University Of Texas System Method and apparatus for programmable fluidic processing
US6841350B2 (en) 1999-02-20 2005-01-11 The North West London Hospitals Nhs Trust Of Northwick Park Hospital Methods of diagnosing prostate cancer through the detection of the presence or absence of Pax 2 mRNA
US6936417B2 (en) 1999-02-22 2005-08-30 Aros Applied Biotechnology Aps Gene expression in bladder tumors
US20040037813A1 (en) 1999-02-25 2004-02-26 Simpson David G. Electroprocessed collagen and tissue engineering
WO2000052455A1 (en) 1999-03-02 2000-09-08 Advion Biosciences, Inc. Integrated monolithic microfabricated dispensing nozzle and liquid chromatography-electrospray system and method
US20040041093A1 (en) 1999-03-02 2004-03-04 Schultz Gary A. Integrated monolithic microfabricated dispensing nozzle and liquid chromatography-electrospray system and method
US6942978B1 (en) 1999-03-03 2005-09-13 The Board Of Trustees Of The University Of Arkansas Transmembrane serine protease overexpressed in ovarian carcinoma and uses thereof
US6171850B1 (en) 1999-03-08 2001-01-09 Caliper Technologies Corp. Integrated devices and systems for performing temperature controlled reactions and analyses
US6428053B1 (en) * 1999-03-12 2002-08-06 California Institute Of Technology Micromachined fluidic coupler and method of making the same
AU3961100A (en) 1999-03-17 2000-10-04 Merck Patent Gmbh Method for producing cosmetic or pharmaceutical formulations by means of a micromixture directly before use
AU768399B2 (en) 1999-03-17 2003-12-11 Merck Patent Gmbh Method for producing cosmetic or pharmaceutical formulations by means of a micromixture directly before use
WO2000054735A1 (en) 1999-03-17 2000-09-21 Merck Patent Gmbh Method for producing cosmetic or pharmaceutical formulations by means of a micromixture directly before use
JP2000271475A (en) 1999-03-23 2000-10-03 Shinji Katsura Finely controlling method of chemical reaction by fine operation of water-in-oil emulsion
US6174160B1 (en) 1999-03-25 2001-01-16 University Of Washington Staged prevaporizer-premixer
US7153700B1 (en) 1999-03-26 2006-12-26 Dana-Farber Cancer Institute, Inc. Methods and compositions for diagnosing and predicting the behavior of cancer
AU4910300A (en) 1999-04-08 2000-11-14 Bernd Penth Method and device for carrying out chemical and physical processes
WO2000061275A2 (en) 1999-04-08 2000-10-19 Bernd Penth Method and device for carrying out chemical and physical processes
US6267353B1 (en) 1999-04-19 2001-07-31 Pbm, Inc. Self draining valve
US20030207295A1 (en) 1999-04-20 2003-11-06 Kevin Gunderson Detection of nucleic acid reactions on bead arrays
US6454193B1 (en) 1999-04-23 2002-09-24 Battellepharma, Inc. High mass transfer electrosprayer
US20010041343A1 (en) 1999-05-04 2001-11-15 Pankowsky Dan A. Products and methods for single parameter and multiparameter phenotyping of cells
US20020179849A1 (en) 1999-05-12 2002-12-05 Kevin Maher Multiplexed fluorescent detection in microfluidic devices
US6506609B1 (en) 1999-05-17 2003-01-14 Caliper Technologies Corp. Focusing of microparticles in microfluidic systems
WO2000070080A1 (en) 1999-05-17 2000-11-23 Caliper Technologies Corp. Focusing of microparticles in microfluidic systems
US6592821B1 (en) 1999-05-17 2003-07-15 Caliper Technologies Corp. Focusing of microparticles in microfluidic systems
US20030229376A1 (en) 1999-06-01 2003-12-11 Biointeractions Ltd. Coated surfaces for immobilizing negatively charged anticoagulating agents from blood fluid
US6738502B1 (en) 1999-06-04 2004-05-18 Kairos Scientific, Inc. Multispectral taxonomic identification
US20060169800A1 (en) 1999-06-11 2006-08-03 Aradigm Corporation Aerosol created by directed flow of fluids and devices and methods for producing same
JP2003501257A (en) 1999-06-11 2003-01-14 アラディジム コーポレーション How to generate an aerosol
WO2000076673A1 (en) 1999-06-11 2000-12-21 Aradigm Corporation Method for producing an aerosol
US6630006B2 (en) 1999-06-18 2003-10-07 The Regents Of The University Of California Method for screening microcrystallizations for crystal formation
US6296673B1 (en) 1999-06-18 2001-10-02 The Regents Of The University Of California Methods and apparatus for performing array microcrystallizations
JP2003502656A (en) 1999-06-22 2003-01-21 テカン トレーディング アーゲー Apparatus and method for performing miniaturized in vitro amplification assays
WO2000078455A1 (en) 1999-06-22 2000-12-28 Tecan Trading Ag Devices and methods for the performance of miniaturized in vitro amplification assays
US6210396B1 (en) 1999-06-24 2001-04-03 Medtronic, Inc. Guiding catheter with tungsten loaded band
US20050226742A1 (en) 1999-06-28 2005-10-13 California Institute Of Technology Microfabricated elastomeric valve and pump systems
US20010029983A1 (en) 1999-06-28 2001-10-18 Unger Marc A. Microfabricated elastomeric valve and pump systems
US6818395B1 (en) 1999-06-28 2004-11-16 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences
US6793753B2 (en) 1999-06-28 2004-09-21 California Institute Of Technology Method of making a microfabricated elastomeric valve
US6408878B2 (en) 1999-06-28 2002-06-25 California Institute Of Technology Microfabricated elastomeric valve and pump systems
US6964847B1 (en) 1999-07-14 2005-11-15 Packard Biosciences Company Derivative nucleic acids and uses thereof
US20010041357A1 (en) 1999-07-28 2001-11-15 Yves Fouillet Method for carrying out a biochemical protocol in continuous flow in a microreactor
WO2001009598A1 (en) 1999-07-28 2001-02-08 University Of Washington Fluidic interconnect, interconnect manifold and microfluidic devices for internal delivery of gases and application of vacuum
US6753147B2 (en) 1999-08-02 2004-06-22 The Johns Hopkins University Digital amplification
US20080241830A1 (en) 1999-08-02 2008-10-02 The Johns Hopkins University Digital amplification
US6440706B1 (en) 1999-08-02 2002-08-27 Johns Hopkins University Digital amplification
US7824889B2 (en) 1999-08-02 2010-11-02 The Johns Hopkins University Digital amplification
US7915015B2 (en) 1999-08-02 2011-03-29 The Johns Hopkins University Digital amplification
US6524456B1 (en) 1999-08-12 2003-02-25 Ut-Battelle, Llc Microfluidic devices for the controlled manipulation of small volumes
WO2001012327A1 (en) 1999-08-12 2001-02-22 Ut-Battelle, Llc Microfluidic devices for the controlled manipulation of small volumes
WO2001014589A2 (en) 1999-08-20 2001-03-01 Luminex Corporation Liquid array technology
US7163801B2 (en) 1999-09-01 2007-01-16 The Burnham Institute Methods for determining the prognosis for cancer patients using tucan
US6439103B1 (en) 1999-09-07 2002-08-27 Vector Engineering Co. Hydraulic and pneumatic cylinder construction
WO2001018244A2 (en) 1999-09-08 2001-03-15 Medical Research Council Selection system
US7090983B1 (en) 1999-09-10 2006-08-15 Takashi Muramatsu Methods for detecting early cancer
US6274320B1 (en) 1999-09-16 2001-08-14 Curagen Corporation Method of sequencing a nucleic acid
US6440760B1 (en) 1999-09-18 2002-08-27 Samsung Electronics Co., Ltd. Method of measuring etched state of semiconductor wafer using optical impedence measurement
US20010050881A1 (en) 1999-09-20 2001-12-13 Depaoli David W. Continuous flow, electrohydrodynamic micromixing apparatus and methods
US6998232B1 (en) 1999-09-27 2006-02-14 Quark Biotech, Inc. Methods of diagnosing bladder cancer
US6890487B1 (en) 1999-09-30 2005-05-10 Science & Technology Corporation ©UNM Flow cytometry for high throughput screening
US6481648B1 (en) 1999-10-01 2002-11-19 Agilent Technologies, Inc. Spray tip for a microfluidic laboratory microchip
US7316906B2 (en) 1999-10-08 2008-01-08 The Feinstein Institute For Medical Research CD38 as a prognostic indicator in B cell chronic lymphocytic leukemia
US7393634B1 (en) 1999-10-12 2008-07-01 United States Of America As Represented By The Secretary Of The Air Force Screening for disease susceptibility by genotyping the CCR5 and CCR2 genes
US7507541B2 (en) 1999-10-28 2009-03-24 Agensys, Inc. 36P6D5: secreted tumor antigen
US7326529B2 (en) 1999-12-06 2008-02-05 Diadexus, Inc. Method of diagnosing, monitoring, staging, imaging and treating prostate cancer
DE19961257A1 (en) 1999-12-18 2001-07-05 Inst Mikrotechnik Mainz Gmbh Micromixer
US20030039169A1 (en) 1999-12-18 2003-02-27 Wolfgang Ehrfeld Micromixer
US7510707B2 (en) 1999-12-20 2009-03-31 New York University Mt. Sinai School Of Medicine PAR, a novel marker gene for breast and prostate cancers
US6540395B2 (en) 1999-12-23 2003-04-01 Ernst Mühlbauer KG Dynamic mixer for dental impression compounds
US20020127591A1 (en) 2000-01-06 2002-09-12 Caliper Technologies Corp. Methods and systems for monitoring intracellular binding reactions
US6790328B2 (en) 2000-01-12 2004-09-14 Ut-Battelle, Llc Microfluidic device and method for focusing, segmenting, and dispensing of a fluid stream
US20020008028A1 (en) 2000-01-12 2002-01-24 Jacobson Stephen C. Microfluidic device and method for focusing, segmenting, and dispensing of a fluid stream
US7314721B2 (en) 2000-01-21 2008-01-01 Ludwig Institute For Cancer Research Small cell lung cancer associated antigens and uses therefor
US20010032053A1 (en) 2000-01-24 2001-10-18 Hielscher Andreas H. Imaging of a scattering medium using the equation of radiative transfer
US20010041344A1 (en) 2000-02-03 2001-11-15 Nanoscale Combinatorial Synthesis, Inc., 625 Clyde Avenue, Mountain View, Ca 94043 Nonredundant split/pool synthesis of combinatorial libraries
US6530944B2 (en) 2000-02-08 2003-03-11 Rice University Optically-active nanoparticles for use in therapeutic and diagnostic methods
US6355193B1 (en) 2000-03-01 2002-03-12 Gale Stott Method for making a faux stone concrete panel
WO2001064332A1 (en) 2000-03-02 2001-09-07 Newcastle Universtiy Ventures Limited Capillary reactor distribution device and method
WO2001068257A1 (en) 2000-03-10 2001-09-20 Bioprocessors Corporation Microreactor
WO2001069289A2 (en) 2000-03-10 2001-09-20 Flow Focusing, Inc. Methods for producing optical fiber by focusing high viscosity liquid
US6808382B2 (en) 2000-03-15 2004-10-26 Lino Lanfranchi Device for conveying and checking containers, in particular preforms
US20010024790A1 (en) 2000-03-17 2001-09-27 Hitachi, Ltd. DNA base sequencing system
US20020012971A1 (en) 2000-03-20 2002-01-31 Mehta Tammy Burd PCR compatible nucleic acid sieving medium
US7229760B2 (en) 2000-03-24 2007-06-12 Micromet Ag mRNA amplification
US6565010B2 (en) 2000-03-24 2003-05-20 Praxair Technology, Inc. Hot gas atomization
US6767704B2 (en) 2000-03-27 2004-07-27 Thomas Jefferson University Methods of screening and diagnosing esophageal cancer by determining guanylin cyclase C expression
US7479376B2 (en) 2000-03-27 2009-01-20 Thomas Jefferson University Compositions and methods for identifying and targeting cancer cells of alimentary canal origin
US7323309B2 (en) 2000-03-28 2008-01-29 Northwestern University Bio-barcodes based on oligonucleotide-modified particles
WO2001072431A1 (en) 2000-03-28 2001-10-04 Nisco Engineering Ag Method and device for producing drops of equal size
DE10015109A1 (en) 2000-03-28 2001-10-04 Peter Walzel Processes and devices for producing drops of equal size
US6409832B2 (en) 2000-03-31 2002-06-25 Micronics, Inc. Protein crystallization in microfluidic structures
US20020065609A1 (en) 2000-04-10 2002-05-30 Matthew Ashby Methods for the survey and genetic analysis of populations
US20020166582A1 (en) 2000-04-14 2002-11-14 Nanostream, Inc. Microfluidic branch metering systems and methods
WO2001080283A1 (en) 2000-04-18 2001-10-25 Waters Investments Limited Improved electrospray and other lc/ms interfaces
JP2001301154A (en) 2000-04-20 2001-10-30 Dainippon Printing Co Ltd Method of attaching liquid with surface tension reduced by voltage application by electric field jet
US6469094B1 (en) 2000-04-28 2002-10-22 Xerox Corporation Polymerization processes
US20020022038A1 (en) 2000-05-05 2002-02-21 Bruno Biatry Microcapsules with an aqueous core containing at least one water-soluble cosmetic or dermatological active principle and cosmetic or dermatological compositions containing them
US20030040620A1 (en) 2000-05-20 2003-02-27 Langmore John P. Method of producing a DNA library using positional amplification
US20050248066A1 (en) 2000-05-22 2005-11-10 Esteban Miguel A S Outer surfaces of sun visors
US20010048900A1 (en) 2000-05-24 2001-12-06 Bardell Ronald L. Jet vortex mixer
US7267938B2 (en) 2000-05-25 2007-09-11 President And Fellows Of Harvard College Patterning of surfaces utilizing microfluidic stamps including three-dimensionally arrayed channel networks
US6645432B1 (en) 2000-05-25 2003-11-11 President & Fellows Of Harvard College Microfluidic systems including three-dimensionally arrayed channel networks
WO2001089787A2 (en) 2000-05-25 2001-11-29 President And Fellows Of Harvard College Microfluidic systems including three-dimensionally arrayed channel networks
WO2001089788A2 (en) 2000-05-25 2001-11-29 President And Fellows Of Harvard College Patterning of surfaces utilizing microfluidic stamps including three-dimensionally arrayed channel networks
US20020004532A1 (en) 2000-05-26 2002-01-10 Michelle Matathia Low emulsifier multiple emulsions
US6660252B2 (en) 2000-05-26 2003-12-09 Color Access, Inc. Low emulsifier multiple emulsions
US6729561B2 (en) 2000-05-30 2004-05-04 Dainippon Screen Mfg. Co., Ltd. Cleaning nozzle and substrate cleaning apparatus
US6680178B2 (en) 2000-06-02 2004-01-20 The Regents Of The University Of California Profiling of protease specificity using combinatorial fluorogenic substrate libraries
US20060263888A1 (en) 2000-06-02 2006-11-23 Honeywell International Inc. Differential white blood count on a disposable card
US7622081B2 (en) 2000-06-05 2009-11-24 California Institute Of Technology Integrated active flux microfluidic devices and methods
WO2001094635A2 (en) 2000-06-05 2001-12-13 California Institute Of Technology Integrated active flux microfluidic devices and methods
US7049072B2 (en) 2000-06-05 2006-05-23 University Of South Florida Gene expression analysis of pluri-differentiated mesenchymal progenitor cells and methods for diagnosing a leukemic disease state
US6974667B2 (en) 2000-06-14 2005-12-13 Gene Logic, Inc. Gene expression profiles in liver cancer
US20030061687A1 (en) 2000-06-27 2003-04-03 California Institute Of Technology, A California Corporation High throughput screening of crystallization materials
US6592321B2 (en) 2000-08-03 2003-07-15 Demag Cranes & Components Gmbh Control and guiding device for manually operating a handling unit, and modular construction kit for making such devices of different configuration
US7081192B1 (en) 2000-08-08 2006-07-25 Aviva Biosciences Corporation Methods for manipulating moieties in microfluidic systems
US20020041378A1 (en) 2000-08-08 2002-04-11 International Business Machines Corporation Polarized light fluorescence imageing device
US20040005582A1 (en) 2000-08-10 2004-01-08 Nanobiodynamics, Incorporated Biospecific desorption microflow systems and methods for studying biospecific interactions and their modulators
US6301055B1 (en) 2000-08-16 2001-10-09 California Institute Of Technology Solid immersion lens structures and methods for producing solid immersion lens structures
US6560030B2 (en) 2000-08-16 2003-05-06 California Institute Of Technology Solid immersion lens structures and methods for producing solid immersion lens structures
US6608726B2 (en) 2000-08-16 2003-08-19 California Institute Of Technology Solid immersion lens structures and methods for producing solid immersion lens structures
US6682890B2 (en) 2000-08-17 2004-01-27 Protein Design Labs, Inc. Methods of diagnosing and determining prognosis of colorectal cancer
WO2002016017A2 (en) 2000-08-25 2002-02-28 INSTITUT FüR MIKROTECHNIK MAINZ GMBH Method and statistical micromixer for mixing at least two liquids
US20040027915A1 (en) 2000-08-25 2004-02-12 Holger Lowe Method and statistical micromixer for mixing at least two liquids
DE10041823A1 (en) 2000-08-25 2002-03-14 Inst Mikrotechnik Mainz Gmbh Method and static micromixer for mixing at least two fluids
US6935768B2 (en) 2000-08-25 2005-08-30 Institut Fur Mikrotechnik Mainz Gmbh Method and statistical micromixer for mixing at least two liquids
US20030148273A1 (en) 2000-08-26 2003-08-07 Shoulian Dong Target enrichment and amplification
US20020106667A1 (en) 2000-08-31 2002-08-08 Nobuko Yamamoto Screening method for gene variation
US6610499B1 (en) 2000-08-31 2003-08-26 The Regents Of The University Of California Capillary array and related methods
WO2002018949A2 (en) 2000-08-31 2002-03-07 The Regents Of The University Of California Capillary array and related methods
JP2002085961A (en) 2000-09-13 2002-03-26 Inst Of Physical & Chemical Res REACTOR AND ITS MANUFACTURING METHOD
WO2002022869A2 (en) 2000-09-13 2002-03-21 Medical Research Council Directed evolution method
US20040005594A1 (en) 2000-09-13 2004-01-08 Medical Research Council Directed evolution method
US20080166772A1 (en) 2000-09-13 2008-07-10 Phillip Hollinger Methods of increasing the concentration of a nucleic acid
US7514210B2 (en) 2000-09-13 2009-04-07 Medical Research Council Compartmentalised self replication method for in vitro evolution of molecular libraries
US6739036B2 (en) 2000-09-13 2004-05-25 Fuji Machine Mfg., Co., Ltd. Electric-component mounting system
US8252539B2 (en) 2000-09-15 2012-08-28 California Institute Of Technology Microfabricated crossflow devices and methods
US20020058332A1 (en) * 2000-09-15 2002-05-16 California Institute Of Technology Microfabricated crossflow devices and methods
US7294503B2 (en) 2000-09-15 2007-11-13 California Institute Of Technology Microfabricated crossflow devices and methods
WO2002023163A1 (en) 2000-09-15 2002-03-21 California Institute Of Technology Microfabricated crossflow devices and methods
US20020085961A1 (en) 2000-09-15 2002-07-04 Morin Jean X. Procedure and apparatus for the cleaning of flue gases containing sulfur dioxide
US20050100895A1 (en) 2000-09-18 2005-05-12 Waldman Scott A. Compositions and methods for identifying and targeting stomach and esophageal cancer cells
WO2002027660A2 (en) 2000-09-29 2002-04-04 Koninklijke Philips Electronics N.V. Image registration system and method using cross-entropy optimization
US6508988B1 (en) 2000-10-03 2003-01-21 California Institute Of Technology Combinatorial synthesis system
WO2002031203A2 (en) 2000-10-10 2002-04-18 Diversa Corporation High throughput or capillary-based screening for a bioactivity or biomolecule
US20030059764A1 (en) 2000-10-18 2003-03-27 Ilya Ravkin Multiplexed cell analysis system
US20020067800A1 (en) 2000-10-19 2002-06-06 Janet Newman Apparatus and method for identification of crystals by in-situ X-ray diffraction
US6450139B1 (en) 2000-10-20 2002-09-17 Unisia Jecs Corporation Valve timing control system for internal combustion engine
WO2002036815A2 (en) 2000-10-28 2002-05-10 Ncimb Ltd. Genetic analysis of microorganisms
US7378280B2 (en) 2000-11-16 2008-05-27 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
US7068874B2 (en) 2000-11-28 2006-06-27 The Regents Of The University Of California Microfluidic sorting device
US6905844B2 (en) 2000-11-28 2005-06-14 Kim Jin-Woo Human cervical cancer 2 protooncogene and protein encoded therein
US20030224509A1 (en) 2000-11-29 2003-12-04 Cangen International DAP-kinase and HOXA9, two human genes associated with genesis, progression, and aggressiveness of non-small cell lung cancer
US20020090720A1 (en) 2000-11-29 2002-07-11 Mutz Mitchell W. Focused acoustic ejection cell sorting system and method
US20100213628A1 (en) 2000-12-07 2010-08-26 President And Fellows Of Harvard College Methods and compositions for encapsulating active agents
WO2002047665A2 (en) 2000-12-07 2002-06-20 President And Fellows Of Harvard College Methods and compositions for encapsulating active agents
US6767194B2 (en) 2001-01-08 2004-07-27 President And Fellows Of Harvard College Valves and pumps for microfluidic systems and method for making microfluidic systems
US7368255B2 (en) 2001-01-29 2008-05-06 Suk-Chul Bae RUNX3 gene showing anti-tumor activity and use thereof
WO2002060591A1 (en) 2001-01-31 2002-08-08 Universidad De Sevilla Device and method for producing stationary multi-component liquid capillary streams and micrometric and nanometric sized capsules
WO2002060275A1 (en) 2001-01-31 2002-08-08 Kraft Foods Holdings, Inc. Production of capsules and particles for improvement of food products
US7501244B2 (en) 2001-02-21 2009-03-10 Novartis Vaccines And Diagnostics, Inc. Determining prognosis of colon or breast cancer by measuring TTK expression
EP1447127A1 (en) 2001-02-23 2004-08-18 Japan Science and Technology Agency Process and apparatus for producing emulsion and microcapsules
US20040068019A1 (en) 2001-02-23 2004-04-08 Toshiro Higuchi Process for producing emulsion and microcapsules and apparatus therefor
WO2002066992A1 (en) 2001-02-23 2002-08-29 Japan Science And Technology Corporation Small liquid particle handling method, and device therefor
US20040134854A1 (en) 2001-02-23 2004-07-15 Toshiro Higuchi Small liquid particle handling method, and device therefor
US7268167B2 (en) 2001-02-23 2007-09-11 Japan Science And Technology Agency Process for producing emulsion and microcapsules and apparatus therefor
EP1741482A2 (en) 2001-02-23 2007-01-10 Japan Science and Technology Agency Process and apparatus for producing microcapsules
WO2002068104A1 (en) 2001-02-23 2002-09-06 Japan Science And Technology Corporation Process for producing emulsion and microcapsules and apparatus therefor
EP1362634A1 (en) 2001-02-23 2003-11-19 Japan Science and Technology Corporation Process for producing emulsion and microcapsules and apparatus therefor
US20020155080A1 (en) 2001-03-05 2002-10-24 Glenn Robert Wayne Delivery of reactive agents via multiple emulsions for use in shelf stable products
US20040092824A1 (en) 2001-03-06 2004-05-13 Stamnes Jakob J. Method and an arrangement for the determination of the optical properties of a multi-layered tissue
US20040037739A1 (en) 2001-03-09 2004-02-26 Mcneely Michael Method and system for microfluidic interfacing to arrays
US20020164629A1 (en) 2001-03-12 2002-11-07 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences by asynchronous base extension
US6717136B2 (en) 2001-03-19 2004-04-06 Gyros Ab Microfludic system (EDI)
US20020143437A1 (en) 2001-03-28 2002-10-03 Kalyan Handique Methods and systems for control of microfluidic devices
WO2002078845A1 (en) 2001-03-28 2002-10-10 Handylab, Inc. Methods and systems for control of microfluidic devices
US20050037397A1 (en) 2001-03-28 2005-02-17 Nanosphere, Inc. Bio-barcode based detection of target analytes
US20060036348A1 (en) 2001-03-28 2006-02-16 Handylab, Inc. Methods and systems for control of microfluidic devices
US20030064414A1 (en) 2001-03-30 2003-04-03 Benecky Michael J. Rapid assessment of coagulation activity in whole blood
US20030008308A1 (en) 2001-04-06 2003-01-09 California Institute Of Technology Nucleic acid amplification utilizing microfluidic devices
US6752922B2 (en) 2001-04-06 2004-06-22 Fluidigm Corporation Microfluidic chromatography
US6960437B2 (en) 2001-04-06 2005-11-01 California Institute Of Technology Nucleic acid amplification utilizing microfluidic devices
US20030082795A1 (en) 2001-04-25 2003-05-01 Michael Shuler Devices and methods for pharmacokinetic-based cell culture system
US20020164271A1 (en) 2001-05-02 2002-11-07 Ho Winston Z. Wavelength-coded bead for bioassay and signature recogniton
US20040259083A1 (en) 2001-05-11 2004-12-23 Mitsuaki Oshima Biomolecular substrate and method and apparatus for examination and diagnosis using the same
US7814175B1 (en) 2001-05-14 2010-10-12 At&T Intellectual Property Ii, L.P. System having generalized client-server computing
US6744046B2 (en) 2001-05-24 2004-06-01 New Objective, Inc. Method and apparatus for feedback controlled electrospray
US20030012586A1 (en) 2001-05-24 2003-01-16 Nobuo Iwata Developer container, developing conveying device and image forming apparatus using the same
US6806058B2 (en) 2001-05-26 2004-10-19 One Cell Systems, Inc. Secretions of proteins by encapsulated cells
US20060147909A1 (en) 2001-05-31 2006-07-06 Markus Rarbach Microstructures and use thereof for the directed evolution of biomolecules
US6797056B2 (en) 2001-06-08 2004-09-28 Syrrx, Inc. Microfluidic method employing delivery of plural different fluids to same lumen
US6872250B2 (en) 2001-06-08 2005-03-29 Syrrx, Inc. Microvolume crystallization method employing multiple lumens
US7171311B2 (en) 2001-06-18 2007-01-30 Rosetta Inpharmatics Llc Methods of assigning treatment to breast cancer patients
US7514209B2 (en) 2001-06-18 2009-04-07 Rosetta Inpharmatics Llc Diagnosis and prognosis of breast cancer patients
US20050003380A1 (en) 2001-06-18 2005-01-06 Medical Research Council Selective gene amplification
WO2002103363A2 (en) 2001-06-18 2002-12-27 Medical Research Council Selection by avidity capture
WO2002103011A2 (en) 2001-06-18 2002-12-27 Medical Research Council Selective gene amplification
US20030015425A1 (en) 2001-06-20 2003-01-23 Coventor Inc. Microfluidic system including a virtual wall fluid interface port for interfacing fluids with the microfluidic system
WO2003003015A3 (en) 2001-06-28 2003-10-09 Advanced Res & Tech Inst Methods of preparing multicolor quantum dot tagged beads and conjugates thereof
US20030148544A1 (en) 2001-06-28 2003-08-07 Advanced Research And Technology Institute, Inc. Methods of preparing multicolor quantum dot tagged beads and conjugates thereof
WO2003003015A2 (en) 2001-06-28 2003-01-09 Advanced Research And Technology Institute, Inc. Methods of preparing multicolor quantum dot tagged beads and conjugates thereof
US6553944B1 (en) 2001-07-03 2003-04-29 Virginia A. Allen Wrist worn leash retaining device
US6656267B2 (en) 2001-07-10 2003-12-02 Structural Genomix, Inc. Tray for macromolecule crystallization and method of using the same
US20030017579A1 (en) 2001-07-10 2003-01-23 Corn Robert M. Surface plasmon resonance imaging of micro-arrays
US20030108900A1 (en) 2001-07-12 2003-06-12 Arnold Oliphant Multiplex nucleic acid reactions
US6557334B2 (en) 2001-07-13 2003-05-06 Willem Jager Caster mounted reel mower
US20030017305A1 (en) 2001-07-17 2003-01-23 Roitman Daniel B. Paek embossing and adhesion for microfluidic devices
US20050048467A1 (en) 2001-07-20 2005-03-03 Sastry Jagannadha K. Method and compositions relating to hpv-associated pre-cancerous and cancerous growths, including cin
US6766817B2 (en) 2001-07-25 2004-07-27 Tubarc Technologies, Llc Fluid conduction utilizing a reversible unsaturated siphon with tubarc porosity action
US6918404B2 (en) 2001-07-25 2005-07-19 Tubarc Technologies, Llc Irrigation and drainage based on hydrodynamic unsaturated fluid flow
US7066586B2 (en) 2001-07-25 2006-06-27 Tubarc Technologies, Llc Ink refill and recharging system
WO2003011443A2 (en) 2001-07-27 2003-02-13 President And Fellows Of Harvard College Laminar mixing apparatus and methods
US20080222741A1 (en) 2001-08-02 2008-09-11 The Regents Of The University Of Michigan Expression Profile Of Prostate Cancer
US7332590B2 (en) 2001-08-16 2008-02-19 The United States Of America As Represented By The Department Of Health And Human Services Molecular characteristics of non-small cell lung cancer
US7741130B2 (en) 2001-08-20 2010-06-22 President And Fellows Of Harvard College Fluidic arrays and method of using
US6520425B1 (en) 2001-08-21 2003-02-18 The University Of Akron Process and apparatus for the production of nanofibers
US20030038865A1 (en) * 2001-08-22 2003-02-27 Shusuke Inamura Ink supply mechanism and inkjet recording apparatus including the ink supply mechanism
US7078180B2 (en) 2001-09-05 2006-07-18 The Children's Hospital Of Philadelphia Methods and compositions useful for diagnosis, staging, and treatment of cancers and tumors
US7390463B2 (en) 2001-09-07 2008-06-24 Corning Incorporated Microcolumn-based, high-throughput microfluidic device
US20050084923A1 (en) 2001-09-14 2005-04-21 Peter-Juergen Mueller Methods for cultivating and analyzing microbial individual cell cultures
US20040241693A1 (en) 2001-09-21 2004-12-02 Florence Ricoul Method for moving a fluid of interest in a capillary tube and fluidic microsystem
WO2003026798A1 (en) 2001-09-21 2003-04-03 Commissariat A L'energie Atomique Method for moving a fluid of interest in a capillary tube and fluidic microsystem
US6429148B1 (en) 2001-10-09 2002-08-06 Promos Technologies, Inc. Anisotropic formation process of oxide layers for vertical transistors
US6670142B2 (en) 2001-10-26 2003-12-30 The Regents Of The University Of California Method for screening combinatorial bead library, capturing cells from body fluids, and ligands for cancer cells
WO2003037302A1 (en) 2001-10-30 2003-05-08 Windsor J Brian Method and system for the co-isolation of cognate dna, rna and protein sequences and method for screening co-isolates for defined activities
US6464336B1 (en) 2001-10-31 2002-10-15 Eastman Kodak Company Ink jet printing with color-balanced ink drops mixed using bleached ink
US20080293578A1 (en) 2001-11-07 2008-11-27 Shaugnessy John D Diagnosis, prognosis and identification of potential therapeutic targets of multiple myeloma based on gene expression profiling
US20080234139A1 (en) 2001-11-07 2008-09-25 Shaughnessy John D Diagnosis, prognosis and identification of potential therapeutic targets of multiple myeloma based on gene expression profiling
US7308364B2 (en) 2001-11-07 2007-12-11 The University Of Arkansas For Medical Sciences Diagnosis of multiple myeloma on gene expression profiling
JP2003149136A (en) 2001-11-13 2003-05-21 Shimadzu Corp Optical image measurement method
US20030143599A1 (en) 2001-11-13 2003-07-31 Rubicon Genomics Inc. DNA amplification and sequencing using DNA molecules generated by random fragmentation
US20040253731A1 (en) 2001-11-16 2004-12-16 Medical Research Council Emulsion compositions
WO2003044187A2 (en) 2001-11-16 2003-05-30 Medical Research Council Emulsion compositions
US20050064460A1 (en) 2001-11-16 2005-03-24 Medical Research Council Emulsion compositions
US7524633B2 (en) 2001-11-16 2009-04-28 The Johns Hopkins University School Of Medicine Method of detection of prostate cancer
US8153402B2 (en) 2001-11-16 2012-04-10 Medical Research Council Emulsion compositions
US7655435B2 (en) 2001-11-16 2010-02-02 Medical Research Council Emulsion compositions
US7429467B2 (en) 2001-11-16 2008-09-30 Medical Research Council Emulsion compositions
US20100159592A1 (en) 2001-11-16 2010-06-24 Phillip Holliger Emulsion compositions
US20070009954A1 (en) 2001-11-28 2007-01-11 Bio-Rad Laboratories, Inc. Parallel polymorphism scoring by amplification and error correction
US7118910B2 (en) 2001-11-30 2006-10-10 Fluidigm Corporation Microfluidic device and methods of using same
US7057026B2 (en) 2001-12-04 2006-06-06 Solexa Limited Labelled nucleotides
US20040096515A1 (en) 2001-12-07 2004-05-20 Bausch Andreas R. Methods and compositions for encapsulating active agents
US20060246431A1 (en) 2001-12-07 2006-11-02 Wamadiva Balachandran Test apparatus
US6800849B2 (en) 2001-12-19 2004-10-05 Sau Lan Tang Staats Microfluidic array devices and methods of manufacture and uses thereof
US20090092973A1 (en) 2001-12-21 2009-04-09 Aviaradx, Inc. Grading of Breast Cancer
US6949342B2 (en) 2001-12-21 2005-09-27 Whitehead Institute For Biomedical Research Prostate cancer diagnosis and outcome prediction by expression analysis
US20030144260A1 (en) 2002-01-03 2003-07-31 Yissum Research Development Company Of The Hebrew University Of Jerusalem Heterocyclic compounds, method of developing new drug leads and combinatorial libraries used in such method
EP1462517A1 (en) 2002-01-08 2004-09-29 Japan Science and Technology Agency Pcr method by electrostatic transportation, hybridization method for electrostatic transportation and devices therefor
US20050079501A1 (en) 2002-01-25 2005-04-14 Hisashi Koike Method and apparatus for detecting nucleic acid data
JP2003222633A (en) 2002-01-30 2003-08-08 Nippon Sheet Glass Co Ltd Microchip
US7341211B2 (en) 2002-02-04 2008-03-11 Universidad De Sevilla Device for the production of capillary jets and micro-and nanometric particles
JP2003232525A (en) 2002-02-08 2003-08-22 Mitsubishi Electric Corp Induction heating cooker
US20030232356A1 (en) 2002-02-08 2003-12-18 Dooley Thomas P. Skin cell biomarkers and methods for identifying biomarkers using nucleic acid microarrays
US20030181574A1 (en) 2002-02-11 2003-09-25 Herve Adam Method for controlling the stability of emulsions
US20040188254A1 (en) 2002-03-05 2004-09-30 Caliper Technologies Corp. Mixed mode microfluidic systems
US20030230486A1 (en) 2002-03-05 2003-12-18 Caliper Technologies Corp. Mixed mode microfluidic systems
US7081340B2 (en) 2002-03-13 2006-07-25 Genomic Health, Inc. Gene expression profiling in biopsied tumor tissues
US20050202429A1 (en) 2002-03-20 2005-09-15 Innovativebio.Biz Microcapsules with controlable permeability encapsulating a nucleic acid amplification reaction mixture and their use as reaction compartment for parallels reactions
WO2003078659A2 (en) 2002-03-20 2003-09-25 Innovativebio.Biz Microcapsules with controlable permeability encapsulating a nucleic acid amplification reaction mixture and their use as reaction compartments for parallels reactions
US7348142B2 (en) 2002-03-29 2008-03-25 Veridex, Lcc Cancer diagnostic panel
US20040229349A1 (en) 2002-04-01 2004-11-18 Fluidigm Corporation Microfluidic particle-analysis systems
US20030183525A1 (en) 2002-04-01 2003-10-02 Xerox Corporation Apparatus and method for using electrostatic force to cause fluid movement
US7300765B2 (en) 2002-04-02 2007-11-27 Ucb Pharma S.A. SC6 for diagnosis of cancers
US7479371B2 (en) 2002-04-09 2009-01-20 Tokai University Method of judging leukemia, pre-leukemia or aleukemic malignant blood disease and diagnostic therefor
US20050183995A1 (en) 2002-04-17 2005-08-25 Cytonome, Inc. Method and apparatus for sorting particles
US20050169797A1 (en) 2002-05-08 2005-08-04 Mitsuaki Oshima Biomolecular substrate, method of testing or diagnosis with use thereof and apparatus therefor
US20110174622A1 (en) 2002-05-09 2011-07-21 The University Of Chicago Device and method for pressure-driven plug transport
US20050087122A1 (en) 2002-05-09 2005-04-28 Ismagliov Rustem F. Device and method for pressure-driven plug transport and reaction
US7901939B2 (en) 2002-05-09 2011-03-08 University Of Chicago Method for performing crystallization and reactions in pressure-driven fluid plugs
US20110142734A1 (en) 2002-05-09 2011-06-16 The University Of Chicago Device and method for pressure-driven plug transport
US20110176966A1 (en) 2002-05-09 2011-07-21 The University Of Chicago Device and method for pressure-driven plug transport
US20110177609A1 (en) 2002-05-09 2011-07-21 The University Of Chicago Device and method for pressure-driven plug transport
WO2004038363A2 (en) 2002-05-09 2004-05-06 The University Of Chicago Microfluidic device and method for pressure-driven plug transport and reaction
US20100233026A1 (en) 2002-05-09 2010-09-16 Ismagliov Rustem F Device and method for pressure-driven plug transport and reaction
US20110177586A1 (en) 2002-05-09 2011-07-21 The University Of Chicago Device and method for pressure-driven plug transport
US20110177494A1 (en) 2002-05-09 2011-07-21 The University Of Chicago Device and method for pressure-driven plug transport
US20050272159A1 (en) 2002-05-09 2005-12-08 Ismagilov Rustem F Device and method for pressure-driven plug transport and reaction
US7129091B2 (en) 2002-05-09 2006-10-31 University Of Chicago Device and method for pressure-driven plug transport and reaction
WO2003099843A2 (en) 2002-05-20 2003-12-04 Dow Corning Corporation Peptide derivatives, and their use for the synthesis of silicon-based composite materials
US20040018525A1 (en) 2002-05-21 2004-01-29 Bayer Aktiengesellschaft Methods and compositions for the prediction, diagnosis, prognosis, prevention and treatment of malignant neoplasma
US20030219754A1 (en) 2002-05-23 2003-11-27 Oleksy Jerome E. Fluorescence polarization detection of nucleic acids
US20060096923A1 (en) 2002-05-24 2006-05-11 Fraunhoffer-Gesellschaft Zur Forderung Der Angewan Method for transferring heterogeneous liquids in microchannels without the occurrence of mixing
US7476506B2 (en) 2002-06-03 2009-01-13 Novartis Vaccines And Diagnostics, Inc. Use of NRG4, or inhibitors thereof, in the treatment of colon and pancreatic cancers
US7198899B2 (en) 2002-06-03 2007-04-03 Chiron Corporation Use of NRG4, or inhibitors thereof, in the treatment of colon and pancreatic cancers
US20040030255A1 (en) 2002-06-05 2004-02-12 Research Foundation Of City University Hybrid-dual-Fourier tomographic algorithm for a fast three-dimensional optical image reconstruction in turbid media
US20040258203A1 (en) 2002-06-17 2004-12-23 Akihito Yamano Crystal evaluating device
US20050008592A1 (en) 2002-06-26 2005-01-13 Nadia Gardel Water-in-oil emulsion foundation
WO2004002627A2 (en) 2002-06-28 2004-01-08 President And Fellows Of Harvard College Method and apparatus for fluid dispersion
US20050172476A1 (en) 2002-06-28 2005-08-11 President And Fellows Of Havard College Method and apparatus for fluid dispersion
US20100172803A1 (en) 2002-06-28 2010-07-08 President And Fellows Of Harvard College Method and apparatus for fluid dispersion
US20050019776A1 (en) 2002-06-28 2005-01-27 Callow Matthew James Universal selective genome amplification and universal genotyping system
US7708949B2 (en) 2002-06-28 2010-05-04 President And Fellows Of Harvard College Method and apparatus for fluid dispersion
US8337778B2 (en) 2002-06-28 2012-12-25 President And Fellows Of Harvard College Method and apparatus for fluid dispersion
US20050161669A1 (en) 2002-08-02 2005-07-28 Jovanovich Stevan B. Integrated system with modular microfluidic components
US20040050946A1 (en) 2002-08-06 2004-03-18 Clean Earth Technologies, Llc Method and apparatus for electrostatic spray
US20070166705A1 (en) 2002-08-23 2007-07-19 John Milton Modified nucleotides
WO2004018497A2 (en) 2002-08-23 2004-03-04 Solexa Limited Modified nucleotides for polynucleotide sequencing
US20060105170A1 (en) 2002-08-29 2006-05-18 Isis Innovation Limited Magnetic particle and process for preparation
WO2004026453A2 (en) 2002-09-06 2004-04-01 Genteric, Inc. Microcapsules and methods of use
US20060003347A1 (en) 2002-09-11 2006-01-05 Medical Research Council Single-molecule in vitro evolution
WO2004024917A2 (en) 2002-09-11 2004-03-25 Medical Research Council Single-molecule in vitro evolution
US20050208495A1 (en) 2002-09-17 2005-09-22 Joseph Richard A Real-time detection of nucleic acid reactions
US20040079881A1 (en) 2002-09-18 2004-04-29 Fischer Steven M. Multimode ionization source
US20040058450A1 (en) 2002-09-24 2004-03-25 Pamula Vamsee K. Methods and apparatus for manipulating droplets by electrowetting-based techniques
US20040057906A1 (en) 2002-09-24 2004-03-25 Li-Chien Hsu Perfluorocarbon emulsions with non-fluorinated surfactants
US20070045117A1 (en) 2002-09-24 2007-03-01 Duke University Apparatuses for mixing droplets
US20070031829A1 (en) 2002-09-30 2007-02-08 Hideyuki Yasuno Oligonucleotides for genotyping thymidylate synthase gene
US20040071781A1 (en) 2002-10-11 2004-04-15 Ferro Corporation Composite particles and method for preparing
WO2004037374A2 (en) 2002-10-23 2004-05-06 The Trustees Of Princeton University Method for continuous particle separation using obstacle arrays asymmetrically aligned to fields
US20040136497A1 (en) 2002-10-30 2004-07-15 Meldrum Deirdre R Preparation of samples and sample evaluation
US20040181343A1 (en) 2002-11-01 2004-09-16 Cellectricon Ab Computer program products and systems for rapidly changing the solution environment around sensors
US20040086892A1 (en) 2002-11-06 2004-05-06 Crothers Donald M. Universal tag assay
US20060108012A1 (en) 2002-11-14 2006-05-25 Barrow David A Microfluidic device and methods for construction and application
US7527933B2 (en) 2002-11-22 2009-05-05 Ganymed Pharmaceuticals Ag Genetic products differentially expressed in tumors and the use thereof
WO2004074504A2 (en) 2002-11-26 2004-09-02 Cornell Research Foundation, Inc. Fluorescent silica-based nanoparticles
US20040101822A1 (en) 2002-11-26 2004-05-27 Ulrich Wiesner Fluorescent silica-based nanoparticles
US20060035386A1 (en) 2002-12-02 2006-02-16 Nec Corporation Fine particle handling unit, chip and sensor mounted with same, and methods for separating, capturing and sensing protein
US20090118158A1 (en) 2002-12-17 2009-05-07 Nastech Pharmaceutical Company Inc. Compositions for enhanced epithelial permeation of peptide yy for treating obesity
US20060223127A1 (en) 2002-12-18 2006-10-05 Ciphergen Biosystems, Inc. Serum biomarkers in lung cancer
US7541383B2 (en) 2002-12-20 2009-06-02 Amgen Inc. Asthma and allergic inflammation modulators
US20040224325A1 (en) 2002-12-20 2004-11-11 Caliper Life Sciences, Inc. Single molecule amplification and detection of DNA
US20050042639A1 (en) 2002-12-20 2005-02-24 Caliper Life Sciences, Inc. Single molecule amplification and detection of DNA length
WO2004083443A1 (en) 2002-12-20 2004-09-30 Caliper Life Sciences, Inc. Single molecule amplification and detection of dna
US20100129896A1 (en) 2002-12-20 2010-05-27 Caliper Life Sciences, Inc. System for differentiating the lengths of nucleic acids of interest in a sample
US20090060797A1 (en) 2002-12-30 2009-03-05 The Regents Of The University Of California Fluid control structures in microfluidic devices
US20060258841A1 (en) 2003-01-17 2006-11-16 Josef Michl Pancreatic cancer associated antigen, antibody thereto, and diagnostic and treatment methods
US20040142329A1 (en) 2003-01-17 2004-07-22 Ingeneus Corporation Probe conjugation to increase multiplex binding motif preference
US20040146866A1 (en) 2003-01-21 2004-07-29 Guoliang Fu Quantitative multiplex detection of nucleic acids
US20040146921A1 (en) 2003-01-24 2004-07-29 Bayer Pharmaceuticals Corporation Expression profiles for colon cancer and methods of use
US6832787B1 (en) 2003-01-24 2004-12-21 Sandia National Laboratories Edge compression manifold apparatus
WO2005003375A2 (en) 2003-01-29 2005-01-13 454 Corporation Methods of amplifying and sequencing nucleic acids
US20050130173A1 (en) 2003-01-29 2005-06-16 Leamon John H. Methods of amplifying and sequencing nucleic acids
US20060040297A1 (en) * 2003-01-29 2006-02-23 Leamon John H Methods of amplifying and sequencing nucleic acids
WO2004070005A2 (en) 2003-01-29 2004-08-19 454 Corporation Double ended sequencing
US20050079510A1 (en) 2003-01-29 2005-04-14 Jan Berka Bead emulsion nucleic acid amplification
US7323305B2 (en) 2003-01-29 2008-01-29 454 Life Sciences Corporation Methods of amplifying and sequencing nucleic acids
US20040185484A1 (en) 2003-01-29 2004-09-23 Costa Gina L. Method for preparing single-stranded DNA libraries
WO2004069849A2 (en) 2003-01-29 2004-08-19 454 Corporation Bead emulsion nucleic acid amplification
US20050032240A1 (en) 2003-02-11 2005-02-10 The Regents Of The University Of California Microfluidic devices for controlled viscous shearing and formation of amphiphilic vesicles
WO2004071638A2 (en) 2003-02-11 2004-08-26 Regents Of The University Of California, The Microfluidic devices and method for controlled viscous shearing and formation of amphiphilic vesicles
US7595195B2 (en) 2003-02-11 2009-09-29 The Regents Of The University Of California Microfluidic devices for controlled viscous shearing and formation of amphiphilic vesicles
US7361474B2 (en) 2003-02-24 2008-04-22 United States Of America As Represented By The Department Of Veterans Affairs Serum macrophage migration inhibitory factor (MIF) as marker for prostate cancer
US20040181131A1 (en) 2003-02-25 2004-09-16 Maynard John D. Determination of pH including hemoglobin correction
US20050170431A1 (en) 2003-02-28 2005-08-04 Plexxikon, Inc. PYK2 crystal structure and uses
US20050123937A1 (en) 2003-03-07 2005-06-09 Thorp H. H. Methods for the electrochemical detection of target compounds
US20040209299A1 (en) 2003-03-07 2004-10-21 Rubicon Genomics, Inc. In vitro DNA immortalization and whole genome amplification using libraries generated from randomly fragmented DNA
US20210088519A1 (en) 2003-03-14 2021-03-25 Bio-Rad Laboratories, Inc. Enzyme quantification
US7041481B2 (en) 2003-03-14 2006-05-09 The Regents Of The University Of California Chemical amplification based on fluid partitioning
US20040180346A1 (en) 2003-03-14 2004-09-16 The Regents Of The University Of California. Chemical amplification based on fluid partitioning
USRE41780E1 (en) 2003-03-14 2010-09-28 Lawrence Livermore National Security, Llc Chemical amplification based on fluid partitioning in an immiscible liquid
US11187702B2 (en) 2003-03-14 2021-11-30 Bio-Rad Laboratories, Inc. Enzyme quantification
US20040182712A1 (en) 2003-03-20 2004-09-23 Basol Bulent M. Process and system for eliminating gas bubbles during electrochemical processing
US20050000970A1 (en) 2003-03-25 2005-01-06 Toyota Jidosha Kabushiki Kaisha Gas storage tank and method of manufacturing the same
AU2004225691B2 (en) 2003-03-31 2010-06-24 United Kingdom Research And Innovation Selection by compartmentalised screening
US10151698B2 (en) 2003-03-31 2018-12-11 United Kingdom Research And Innovation Selection by compartmentalized screening
WO2004087308A1 (en) 2003-03-31 2004-10-14 Medical Research Council Method of synthesis and testing of combinatorial libraries using microcapsules
US20190107489A1 (en) 2003-03-31 2019-04-11 Medical Research Council Selection by compartmentalised screening
US20120010107A1 (en) 2003-03-31 2012-01-12 Medical Research Council Selection by compartmentalised screening
US20100210479A1 (en) 2003-03-31 2010-08-19 Medical Research Council Method of synthesis and testing of cominatorial libraries using microcapsules
US20060154298A1 (en) 2003-03-31 2006-07-13 Medical Research Council Method of synthesis and testing of combinatorial libraries using microcapsules
US20120010098A1 (en) 2003-03-31 2012-01-12 Medical Research Council Selection by compartmentalised screening
US20180361346A1 (en) 2003-03-31 2018-12-20 Medical Research Council Method of synthesis and testing of cominatorial libraries using microcapsules
AU2010224352A1 (en) 2003-03-31 2010-10-14 United Kingdom Research And Innovation Selection by compartmentalised screening
US10052605B2 (en) 2003-03-31 2018-08-21 Medical Research Council Method of synthesis and testing of combinatorial libraries using microcapsules
CA2520548A1 (en) 2003-03-31 2004-10-14 Medical Research Council Selection by compartmentalised screening
US9857303B2 (en) 2003-03-31 2018-01-02 Medical Research Council Selection by compartmentalised screening
WO2004088314A1 (en) 2003-03-31 2004-10-14 Medical Research Council Selection by compartmentalised screening
US20060153924A1 (en) 2003-03-31 2006-07-13 Medical Research Council Selection by compartmentalised screening
US9448172B2 (en) 2003-03-31 2016-09-20 Medical Research Council Selection by compartmentalised screening
US7718578B2 (en) 2003-03-31 2010-05-18 Medical Research Council Method of synthesis and testing of combinatorial libraries using microcapsules
US6926313B1 (en) 2003-04-02 2005-08-09 Sandia National Laboratories High pressure capillary connector
WO2004091763A2 (en) 2003-04-10 2004-10-28 President And Fellows Of Harvard College Formation and control of fluidic species
EP2127736A1 (en) 2003-04-10 2009-12-02 The President and Fellows of Harvard College Formation and control of fluidic species
US20060163385A1 (en) 2003-04-10 2006-07-27 Link Darren R Formation and control of fluidic species
US9038919B2 (en) 2003-04-10 2015-05-26 President And Fellows Of Harvard College Formation and control of fluidic species
US7378233B2 (en) 2003-04-12 2008-05-27 The Johns Hopkins University BRAF mutation T1796A in thyroid cancers
US20050095611A1 (en) 2003-05-02 2005-05-05 Chan Daniel W. Identification of biomarkers for detecting pancreatic cancer
US7449303B2 (en) 2003-05-02 2008-11-11 Health Research, Inc. Use of JAG2 expression in diagnosis of plasma cell disorders
US20040224419A1 (en) 2003-05-06 2004-11-11 Thrombodyne, Inc. Systems and methods for measuring fluid properties
WO2004102204A1 (en) 2003-05-16 2004-11-25 Global Technologies (Nz) Ltd Method and apparatus for mixing sample and reagent in a suspension fluid
DE10322893A1 (en) 2003-05-19 2004-12-16 Hans-Knöll-Institut für Naturstoff-Forschung e.V. Equipment for microtechnological structuring of fluids used in analytical or combinatorial biology or chemistry, has dosing, splitting and fusion devices in fluid pathway
WO2004103565A2 (en) 2003-05-19 2004-12-02 Hans-Knöll-Institut für Naturstoff-Forschung e.V. Device and method for structuring liquids and for dosing reaction liquids into liquid compartments immersed in a separation medium
US7156917B2 (en) 2003-05-22 2007-01-02 Hideaki Moriyama Apparatus and method for growing crystal, and apparatus and method for analyzing crystal
US20050129582A1 (en) 2003-06-06 2005-06-16 Micronics, Inc. System and method for heating, cooling and heat cycling on microfluidic device
US20060281089A1 (en) 2003-06-12 2006-12-14 University Of Manitoba Methods for detecting cancer and monitoring cancer progression
US7056674B2 (en) 2003-06-24 2006-06-06 Genomic Health, Inc. Prediction of likelihood of cancer recurrence
JP2005037346A (en) 2003-06-25 2005-02-10 Aisin Seiki Co Ltd Micro fluid control system
US7115230B2 (en) 2003-06-26 2006-10-03 Intel Corporation Hydrodynamic focusing devices
US20060115821A1 (en) 2003-06-26 2006-06-01 Richard Einstein Prostate specific genes and the use thereof as targets for prostate cancer therapy
US20070213410A1 (en) 2003-06-30 2007-09-13 Raustech Pty Ltd. Charged Emulsions For Site-Specific Deposition Of Matter At Micro And Nano Scale
WO2005000970A1 (en) 2003-06-30 2005-01-06 Raustech Pty Ltd Charged emulsions for site-specific deposition of matter at micro and nano scale
WO2005002730A1 (en) 2003-07-02 2005-01-13 The University Of Manchester Microfluidic method and device
US20070065823A1 (en) 2003-07-05 2007-03-22 Devin Dressman Method and compositions for detection and enumeration of genetic variations
WO2005010145A2 (en) 2003-07-05 2005-02-03 The Johns Hopkins University Method and compositions for detection and enumeration of genetic variations
US20070184439A1 (en) 2003-07-17 2007-08-09 Guilford Parry J Markers for detection of gastric cancer
US20080113340A1 (en) 2003-07-18 2008-05-15 Georgetown University Diagnosis and treatment of cervical cancer
US20080206756A1 (en) 2003-07-18 2008-08-28 California Pacific Medical Center Biomarker panel for colorectal cancer
WO2005011867A2 (en) 2003-07-31 2005-02-10 Handylab, Inc. Processing particle-containing samples
US20050032238A1 (en) 2003-08-07 2005-02-10 Nanostream, Inc. Vented microfluidic separation devices and methods
US7473531B1 (en) 2003-08-08 2009-01-06 Colora Corporation Pancreatic cancer targets and uses thereof
US20050048669A1 (en) 2003-08-26 2005-03-03 Nanostream, Inc. Gasketless microfluidic device interface
US20070003442A1 (en) 2003-08-27 2007-01-04 President And Fellows Of Harvard College Electronic control of fluidic species
US8765485B2 (en) 2003-08-27 2014-07-01 President And Fellows Of Harvard College Electronic control of fluidic species
WO2005021151A1 (en) 2003-08-27 2005-03-10 President And Fellows Of Harvard College Electronic control of fluidic species
US20050207940A1 (en) 2003-08-28 2005-09-22 Butler William F Methods and apparatus for sorting cells using an optical switch in a microfluidic channel network
US7622076B2 (en) 2003-09-05 2009-11-24 Stokes Bio Limited Microfluidic analysis system
US10676786B2 (en) 2003-09-05 2020-06-09 Stokes Bio Ltd. Microfluidic analysis system
WO2005023427A1 (en) 2003-09-05 2005-03-17 Stokes Bio Limited A microfluidic analysis system
US20070053896A1 (en) 2003-09-05 2007-03-08 Royal Women's Hospital Diagnostic marker for ovarian cancer
US7479370B2 (en) 2003-09-08 2009-01-20 Health Research, Inc. Detection of 13q14 chromosomal alterations
US20060286570A1 (en) 2003-09-09 2006-12-21 Rowlen Kathy L Use of photopolymerization for amplification and detection of a molecular recognition event
US20050170373A1 (en) 2003-09-10 2005-08-04 Althea Technologies, Inc. Expression profiling using microarrays
US7504214B2 (en) 2003-09-19 2009-03-17 Biotheranostics, Inc. Predicting outcome with tamoxifen in breast cancer
US20100173293A1 (en) 2003-09-19 2010-07-08 Life Technologies Corporation High Density Sequence Detection Methods
WO2005028674A2 (en) 2003-09-22 2005-03-31 Trisogen Biotechnology Limited Partnership Methods and kits useful for detecting an alteration in a locus copy number
US20060269971A1 (en) 2003-09-26 2006-11-30 Mount Sinai Hospital Methods for detecting prostate cancer
US7332280B2 (en) 2003-10-14 2008-02-19 Ronald Levy Classification of patients having diffuse large B-cell lymphoma based upon gene expression
US20050221341A1 (en) 2003-10-22 2005-10-06 Shimkets Richard A Sequence-based karyotyping
WO2005041884A2 (en) 2003-10-31 2005-05-12 Engineered Release Systems, Inc Polymer-based microstructures
US7204431B2 (en) 2003-10-31 2007-04-17 Agilent Technologies, Inc. Electrospray ion source for mass spectroscopy
US20050152908A1 (en) 2003-11-03 2005-07-14 Genenews Inc. Liver cancer biomarkers
US7691576B2 (en) 2003-11-03 2010-04-06 Medical Research Council Compartmentalized self tagging
US20070042400A1 (en) 2003-11-10 2007-02-22 Choi K Y Methods of preparing nucleic acid for detection
US7169560B2 (en) 2003-11-12 2007-01-30 Helicos Biosciences Corporation Short cycle methods for sequencing polynucleotides
US20090191565A1 (en) 2003-11-12 2009-07-30 Helicos Biosciences Corporation Short cycle methods for sequencing polynucleotides
US20050103690A1 (en) 2003-11-19 2005-05-19 Aisin Seiki Kabushiki Kaisha Micro liquid control system
US20070077572A1 (en) 2003-11-24 2007-04-05 Yeda Research And Development Co. Ltd. Compositions and methods for in vitro sorting of molecular and cellular libraries
WO2005049787A2 (en) 2003-11-24 2005-06-02 Yeda Research And Development Co.Ltd. Compositions and methods for in vitro sorting of molecular and cellular libraries
US20050208529A1 (en) 2003-12-01 2005-09-22 Lars Winther Methods and compositions for immuno-histochemical detection
US7736890B2 (en) 2003-12-31 2010-06-15 President And Fellows Of Harvard College Assay device and method
JP2005192944A (en) 2004-01-09 2005-07-21 Keio Gijuku Telemedicine system for pigmentation site
US20050214173A1 (en) 2004-01-25 2005-09-29 Fluidigm Corporation Integrated chip carriers with thermocycler interfaces and methods of using the same
US20060046257A1 (en) 2004-01-27 2006-03-02 Sarah Pollock Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis of lung cancer
US20080014590A1 (en) 2004-01-27 2008-01-17 Compugen Ltd. Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis of prostate cancer
US20050227264A1 (en) * 2004-01-28 2005-10-13 Nobile John R Nucleic acid amplification with continuous flow emulsion
US20080044828A1 (en) 2004-02-04 2008-02-21 Kwok Tim T CUDR as biomarker for cancer progression and therapeutic response
US20060195269A1 (en) 2004-02-25 2006-08-31 Yeatman Timothy J Methods and systems for predicting cancer outcome
US7507532B2 (en) 2004-03-08 2009-03-24 Medigen Biotechnology Corporation Cancer specific gene MH15
US20050202489A1 (en) * 2004-03-12 2005-09-15 Cho Yoon-Kyoung Method and apparatus for amplifying nucleic acids
EP1574586A2 (en) * 2004-03-12 2005-09-14 Samsung Electronics Co., Ltd. Method and apparatus for amplifying nucleic acids
US20080050378A1 (en) 2004-03-23 2008-02-28 Oncotherapy Science, Inc. Method for Diagnosing Non-Small Cell Lung Cancer
US20050260566A1 (en) 2004-03-24 2005-11-24 Tripath Imaging, Inc. Methods and compositions for the detection of cervical disease
US20190024261A1 (en) 2004-03-31 2019-01-24 President And Fellows Of Harvard College Compartmentalised combinatorial chemistry by microfluidic control
US20070184489A1 (en) 2004-03-31 2007-08-09 Medical Research Council Harvard University Compartmentalised combinatorial chemistry by microfluidic control
US20090197772A1 (en) 2004-03-31 2009-08-06 Andrew Griffiths Compartmentalised combinatorial chemistry by microfluidic control
US9925504B2 (en) 2004-03-31 2018-03-27 President And Fellows Of Harvard College Compartmentalised combinatorial chemistry by microfluidic control
US20070092914A1 (en) 2004-03-31 2007-04-26 Medical Research Council, Harvard University Compartmentalised screening by microfluidic control
US20180272299A1 (en) 2004-03-31 2018-09-27 President And Fellows Of Harvard College Compartmentalised combinatorial chemistry by microfluidic control
US20050221339A1 (en) 2004-03-31 2005-10-06 Medical Research Council Harvard University Compartmentalised screening by microfluidic control
US9839890B2 (en) 2004-03-31 2017-12-12 National Science Foundation Compartmentalised combinatorial chemistry by microfluidic control
US20080032413A1 (en) 2004-04-12 2008-02-07 Byeang-Hyean Kim Oligonucleotide For Detecting Target Dna Or Rna
WO2005103106A1 (en) 2004-04-23 2005-11-03 Eugenia Kumacheva Method of producing polymeric particles with selected size, shape, morphology and composition
US7482129B2 (en) 2004-05-04 2009-01-27 Institute Of Virology, Slovak Academy Of Sciences MN/CA IX/CA9 and Renal Cancer Prognosis
US20060008824A1 (en) 2004-05-20 2006-01-12 Leland Stanford Junior University Methods and compositions for clonal amplification of nucleic acid
US20110024455A1 (en) 2004-05-21 2011-02-03 Pepsico, Inc. Beverage dispensing system with a head capable of dispensing plural different beverages
US20070142720A1 (en) 2004-05-24 2007-06-21 Trent Ridder Apparatus and methods for mitigating the effects of foreign interferents on analyte measurements in spectroscopy
US7635562B2 (en) 2004-05-25 2009-12-22 Helicos Biosciences Corporation Methods and devices for nucleic acid sequence determination
US20050287572A1 (en) 2004-06-01 2005-12-29 The Regents Of The University Of California Microfabricated integrated DNA analysis system
WO2005118867A2 (en) 2004-06-01 2005-12-15 The Regents Of The University Of California Microfabricated integrated dna analysis system
WO2005118138A1 (en) 2004-06-04 2005-12-15 Crystal Vision Microsystems Llc Device and process for continuous on-chip flow injection analysis
WO2006009657A1 (en) 2004-06-17 2006-01-26 Essilor International (Compagnie Generale D'optique) Progressive addition lenses with reduced unwanted astigmatism
US20070156037A1 (en) 2004-06-17 2007-07-05 The Regents Of The University Of Californa Time-resolved non-invasive optometric device for medical diagnostic
US20070269804A1 (en) 2004-06-19 2007-11-22 Chondrogene, Inc. Computer system and methods for constructing biological classifiers and uses thereof
US20070154889A1 (en) 2004-06-25 2007-07-05 Veridex, Llc Methods and reagents for the detection of melanoma
US20060003439A1 (en) 2004-07-02 2006-01-05 Ismagilov Rustem F Microfluidic system
WO2006002641A1 (en) 2004-07-02 2006-01-12 Versamatrix A/S Spherical radiofrequency-encoded beads
US20090081685A1 (en) 2004-07-09 2009-03-26 Tripath Imaging, Inc. Methods and compositions for the detection of ovarian disease
US20080038754A1 (en) 2004-07-14 2008-02-14 The Regents Of The University Of California Biomarkers for Early Detection of Ovarian Cancer
US20060100788A1 (en) 2004-07-14 2006-05-11 Invitrogen Corporation Collections of matched biological reagents and methods for identifying matched reagents
US20090023137A1 (en) 2004-07-16 2009-01-22 Oncomethylome Sciences S.A. ESR1 and Cervical Cancer
US20060078475A1 (en) 2004-07-29 2006-04-13 Yu-Chong Tai Modular microfluidic packaging system
US7405002B2 (en) 2004-08-04 2008-07-29 Agency For Science, Technology And Research Coated water-soluble nanoparticles comprising semiconductor core and silica coating
US20060040197A1 (en) 2004-08-20 2006-02-23 Kabushiki Kaisha Toshiba Toner and fixing method
US20060051329A1 (en) 2004-08-27 2006-03-09 The Regents Of The University Of California Microfluidic device for the encapsulation of cells with low and high cell densities
US20080023330A1 (en) 2004-09-09 2008-01-31 Institut Curie Device for Manipulation of Packets in Micro-Containers, in Particular in Microchannels
WO2006027757A2 (en) 2004-09-09 2006-03-16 Institut Curie Microfluidic device using a collinear electric field
US20060068398A1 (en) 2004-09-24 2006-03-30 Cepheid Universal and target specific reagent beads for nucleic acid amplification
US20070120899A1 (en) 2004-09-30 2007-05-31 Ngk Insulators, Ltd. Liquid drop discharge piezoelectric device
US7698287B2 (en) 2004-09-30 2010-04-13 Microsoft Corporation Design of spreadsheet functions for working with tables of data
US20130217601A1 (en) 2004-10-08 2013-08-22 Andrew David Griffiths In vitro evolution in microfluidic systems
US9919277B2 (en) 2004-10-08 2018-03-20 Medical Research Council In vitro evolution in microfluidic systems
US20090197248A1 (en) 2004-10-08 2009-08-06 President And Fellows Of Harvard College Vitro evolution in microfluidic systems
US8871444B2 (en) 2004-10-08 2014-10-28 Medical Research Council In vitro evolution in microfluidic systems
US9925501B2 (en) 2004-10-08 2018-03-27 Medical Research Council In vitro evolution in microfluidic systems
JP2016063824A (en) 2004-10-08 2016-04-28 メディカル リサーチ カウンシルＭｅｄｉｃａｌ Ｒｅｓｅａｒｃｈ Ｃｏｕｎｃｉｌ In vitro evolution in microfluidic systems
US20060078888A1 (en) 2004-10-08 2006-04-13 Medical Research Council Harvard University In vitro evolution in microfluidic systems
US20180272294A1 (en) 2004-10-08 2018-09-27 Medical Research Council In vitro evolution in microfluidic systems
WO2006038035A2 (en) 2004-10-08 2006-04-13 Medical Research Council In vitro evolution in microfluidic systems
US20130157872A1 (en) 2004-10-08 2013-06-20 Andrew David Griffiths In vitro evolution in microfluidic systems
US9186643B2 (en) 2004-10-08 2015-11-17 Medical Research Council In vitro evolution in microfluidic systems
US20130178368A1 (en) 2004-10-08 2013-07-11 Andrew David Griffiths In vitro evolution in microfluidic systems
US10639598B2 (en) 2004-10-08 2020-05-05 President And Fellows Of Harvard College In vitro evolution in microfluidic systems
US9029083B2 (en) 2004-10-08 2015-05-12 Medical Research Council Vitro evolution in microfluidic systems
JP2013143959A (en) 2004-10-08 2013-07-25 Medical Research Council In-vitro evolution in microfluidic system
US7968287B2 (en) 2004-10-08 2011-06-28 Medical Research Council Harvard University In vitro evolution in microfluidic systems
US20060078893A1 (en) 2004-10-12 2006-04-13 Medical Research Council Compartmentalised combinatorial chemistry by microfluidic control
US20090005254A1 (en) 2004-10-12 2009-01-01 Andrew Griffiths Compartmentalized Screening by Microfluidic Control
WO2006040554A1 (en) 2004-10-12 2006-04-20 Medical Research Council Compartmentalised combinatorial chemistry by microfluidic control
WO2006040551A2 (en) 2004-10-12 2006-04-20 Medical Research Council Compartmentalised screening by microfluidic control
WO2006042303A2 (en) 2004-10-12 2006-04-20 Ambion, Inc. Methods and compositions for analyzing ribonucleic acids
US7655470B2 (en) 2004-10-29 2010-02-02 University Of Chicago Method for manipulating a plurality of plugs and performing reactions therein in microfluidic systems
US20060094119A1 (en) 2004-10-29 2006-05-04 Ismagilov Rustem F Microfluidic system
US20060110759A1 (en) 2004-11-05 2006-05-25 Regents Of The University Of California Biomarkers for prostate cancer metastasis
US7416851B2 (en) 2004-11-08 2008-08-26 Institut Pasteur Method of diagnosis/prognosis of human chronic lymphocytic leukemia comprising the profiling of LPL/ADAM genes
US20060234254A1 (en) 2004-11-08 2006-10-19 Sungwhan An Colon cancer biomarker discovery
US20080004436A1 (en) 2004-11-15 2008-01-03 Yeda Research And Development Co. Ltd. At The Weizmann Institute Of Science Directed Evolution and Selection Using in Vitro Compartmentalization
US20060252057A1 (en) 2004-11-30 2006-11-09 Mitch Raponi Lung cancer prognostics
US20070016078A1 (en) 2004-12-06 2007-01-18 Hoyt Clifford C Systems and methods for in-vivo optical imaging and measurement
US20060160762A1 (en) 2004-12-13 2006-07-20 Children's Medical Center Corporation Methods for the treatment, diagnosis, and prognosis of cancer
US20080118462A1 (en) 2005-01-07 2008-05-22 The John Hopkins University Biomarkers for Melanoma
WO2006078841A1 (en) 2005-01-21 2006-07-27 President And Fellows Of Harvard College Systems and methods for forming fluidic droplets encapsulated in particles such as colloidal particles
WO2006076810A1 (en) 2005-01-21 2006-07-27 Perceptronix Medical Inc. Method And Apparatus For Measuring Cancerous Changes From Reflectance Spectral Measurements Obtained During Endoscopic Imaging
US7442507B2 (en) 2005-01-24 2008-10-28 New York University School Of Medicine Methods for detecting circulating mutant BRAF DNA
US20080064047A1 (en) 2005-01-28 2008-03-13 Zetter Bruce R Methods for diagnosis and prognosis of epithelial cancers
US20080003571A1 (en) 2005-02-01 2008-01-03 Mckernan Kevin Reagents, methods, and libraries for bead-based sequencing
US20060177832A1 (en) 2005-02-10 2006-08-10 Sydney Brenner Genetic analysis by sequence-specific sorting
US7393665B2 (en) 2005-02-10 2008-07-01 Population Genetics Technologies Ltd Methods and compositions for tagging and identifying polynucleotides
US20080286199A1 (en) 2005-02-16 2008-11-20 Livingston David M Methods of Detecting Ovarian Cancer
US20080268473A1 (en) 2005-02-17 2008-10-30 Moses Marsha A Adamts-7 as a Biomarker for Cancers of Epithelial Origin
US20060257893A1 (en) * 2005-02-18 2006-11-16 Toru Takahashi Devices and methods for monitoring genomic DNA of organisms
US20080286811A1 (en) 2005-02-18 2008-11-20 Moses Marsha A Cyr61 as a Biomarker for Diagnosis and Prognosis of Cancers of Epithelial Origin
US7604938B2 (en) 2005-02-18 2009-10-20 Canon U.S. Life Sciences, Inc. Devices and methods for monitoring genomic DNA of organisms
US20060234259A1 (en) 2005-02-25 2006-10-19 Rubin Mark A Biomarkers for predicting prostate cancer progression
WO2006096571A2 (en) 2005-03-04 2006-09-14 President And Fellows Of Harvard College Method and apparatus for forming multiple emulsions
US20090009855A1 (en) 2005-03-04 2009-01-08 Japan Science And Technology Agency Wide-band optical amplifier
US20090131543A1 (en) 2005-03-04 2009-05-21 Weitz David A Method and Apparatus for Forming Multiple Emulsions
US20070054119A1 (en) 2005-03-04 2007-03-08 Piotr Garstecki Systems and methods of forming particles
US7510842B2 (en) 2005-03-11 2009-03-31 Vermilllion, Inc. Biomarker for ovarian and endometrial cancer: hepcidin
US7897044B2 (en) 2005-03-11 2011-03-01 Centre National De La Recherche Scientifique Fluid separation device
US20060234264A1 (en) 2005-03-14 2006-10-19 Affymetrix, Inc. Multiplex polynucleotide synthesis
WO2006101851A2 (en) 2005-03-16 2006-09-28 University Of Chicago Microfluidic system
US20060269934A1 (en) 2005-03-16 2006-11-30 Applera Corporation Compositions and methods for clonal amplification and analysis of polynucleotides
US7468271B2 (en) 2005-04-06 2008-12-23 President And Fellows Of Harvard College Molecular characterization with carbon nanotube control
US20060247532A1 (en) 2005-05-02 2006-11-02 Nirmala Ramanujam Method for extraction of optical properties from diffuse reflectance spectra
US8383061B2 (en) 2005-05-02 2013-02-26 Massachusetts Institute Of Technology Microfluidic bubble logic devices
US20060245971A1 (en) 2005-05-02 2006-11-02 Burns Andrew A Photoluminescent silica-based sensors and methods of use
US7473530B2 (en) 2005-05-04 2009-01-06 Wayne State University Method to detect lung cancer
US20080280285A1 (en) 2005-05-11 2008-11-13 Chen Zongyuan G Systems and Methods For Testing using Microfluidic Chips
US20080274513A1 (en) 2005-05-11 2008-11-06 Shenderov Alexander D Method and Device for Conducting Biochemical or Chemical Reactions at Multiple Temperatures
WO2007021343A2 (en) 2005-05-18 2007-02-22 Cornell Research Foundation, Inc. Pharmacokinetic-based culture system with biological barriers
US20090317798A1 (en) 2005-06-02 2009-12-24 Heid Christian A Analysis using microfluidic partitioning devices
US20120253689A1 (en) 2005-06-07 2012-10-04 Rogan Peter K Ab initio generation of single copy genomic probes
US20060281098A1 (en) 2005-06-14 2006-12-14 Xin Miao Method and kits for multiplex hybridization assays
US20070009914A1 (en) 2005-07-07 2007-01-11 Wallace Robert B Labeled complimentary oligonucleotides to detect oligonucleotide-linked ligands
US20070026439A1 (en) 2005-07-15 2007-02-01 Applera Corporation Fluid processing device and method
US20090118128A1 (en) 2005-07-20 2009-05-07 Xiaohai Liu Preparation of templates for nucleic acid sequencing
US20090134027A1 (en) 2005-07-25 2009-05-28 Commissariat A L'energie Atomique Method for Controlling a Communication Between Two Areas By Electrowetting, a Device Including Areas Isolatable From Each Other and Method for making Such a Device
WO2007012638A1 (en) 2005-07-25 2007-02-01 Commissariat A L'energie Atomique Method for controlling communication between two electrowetting zones, device comprising zones capable of being isolated from one another and method for making such a device
US7632562B2 (en) 2005-08-04 2009-12-15 Eastman Kodak Company Universal print media
US7990525B2 (en) 2005-08-08 2011-08-02 Bay Bioscience Kabushiki Kaisha Flow cytometer and flow cytometry
US20070123430A1 (en) 2005-08-18 2007-05-31 David Pasquier Well fluid comprising a fluorinated liquid phase
US10450604B2 (en) 2005-08-22 2019-10-22 Applied Biosystems, Llc Device and method for making discrete volumes of a first fluid in contact with a second fluid, which are immiscible with each other
US9194772B2 (en) 2005-08-22 2015-11-24 Applied Biosystems, Llc Apparatus, system, and method using immiscible-fluid-discrete-volumes
US20070141593A1 (en) 2005-08-22 2007-06-21 Lee Linda G Apparatus, system, and method using immiscible-fluid-discrete-volumes
US20070039866A1 (en) 2005-08-22 2007-02-22 Schroeder Benjamin G Device, system, and method for depositing processed immiscible-fluid-discrete-volumes
US10041113B2 (en) 2005-08-22 2018-08-07 Applied Biosystems, Llc Apparatus, system, and method using immiscible-fluid-discrete-volumes
US20070048744A1 (en) 2005-08-26 2007-03-01 Stanley Lapidus Single molecule sequencing of captured nucleic acids
US7666593B2 (en) 2005-08-26 2010-02-23 Helicos Biosciences Corporation Single molecule sequencing of captured nucleic acids
US20070111303A1 (en) 2005-09-01 2007-05-17 Hiroshi Inoue Method and molecular diagnostic device for detection, analysis and identification of genomic DNA
US20090134331A1 (en) 2005-09-02 2009-05-28 Yuta Miyamae Method of evaluating skin conditions and method of estimating skin thickness
WO2007026884A1 (en) 2005-09-02 2007-03-08 Pola Chemical Industries Inc. Method of evaluating skin conditions and method of estimating skin thickness
US7556776B2 (en) 2005-09-08 2009-07-07 President And Fellows Of Harvard College Microfluidic manipulation of fluids and reactions
WO2007030501A2 (en) 2005-09-08 2007-03-15 Brandeis University Microfluidic manipulation of fluids and reactions
US20070056853A1 (en) 2005-09-15 2007-03-15 Lucnet Technologies Inc. Micro-chemical mixing
JP2007190364A (en) 2005-09-23 2007-08-02 Astron Clinica Ltd Image processing method and apparatus
US20110059435A1 (en) * 2005-10-24 2011-03-10 The John Hopkins University Methods for Beaming
US20080311604A1 (en) 2005-11-02 2008-12-18 Elting James J Methods for Prediction and Prognosis of Cancer, and Monitoring Cancer Therapy
US20120021930A1 (en) 2005-11-22 2012-01-26 Stichting Dienst Landbouwkundig Onderzoek Multiplex Nucleic Acid Detection
US7358231B1 (en) 2005-12-01 2008-04-15 Applera Corporation Pancreatic cancer secreted targets and uses thereof
US20090131353A1 (en) 2005-12-07 2009-05-21 Insel Paul A Diagnosis and Treatment of Chronic Lymphocytic Leukemia (CLL)
US20080299565A1 (en) 2005-12-12 2008-12-04 Schneider Thomas D Probe for Nucleic Acid Sequencing and Methods of Use
US20090075311A1 (en) 2005-12-21 2009-03-19 Johann Karl Assessing colorectal cancer by measuring hemoglobin and m2-pk in a stool sample
US20080286801A1 (en) 2005-12-21 2008-11-20 Carlos Buesa Arjol Method for the analysis of differential expression in colorectal cancer
US9410151B2 (en) 2006-01-11 2016-08-09 Raindance Technologies, Inc. Microfluidic devices and methods of use in the formation and control of nanoreactors
US20140323317A1 (en) 2006-01-11 2014-10-30 Raindance Technologies, Inc. Microfluidic devices and methods of use in the formation and control of nanoreactors
WO2007081385A2 (en) 2006-01-11 2007-07-19 Raindance Technologies, Inc. Microfluidic devices and methods of use in the formation and control of nanoreactors
US20130217583A1 (en) 2006-01-11 2013-08-22 Darren Link Microfluidic devices and methods of use in the formation and control of nanoreactors
US20180355350A1 (en) 2006-01-11 2018-12-13 Raindance Technologies, Inc. Microfluidic devices and methods of use in the formation and control of nanoreactors
US20140256595A1 (en) 2006-01-11 2014-09-11 Raindance Technologies, Inc. Microfluidic devices and methods of use in the formation and control of nanoreactors
US10633652B2 (en) 2006-01-11 2020-04-28 Bio-Rad Laboratories, Inc. Microfluidic devices and methods of use in the formation and control of nanoreactors
US9534216B2 (en) 2006-01-11 2017-01-03 Raindance Technologies, Inc. Microfluidic devices and methods of use in the formation and control of nanoreactors
EP2363205A3 (en) 2006-01-11 2014-06-04 Raindance Technologies, Inc. Microfluidic Devices And Methods Of Use In The Formation And Control Of Nanoreactors
WO2007081387A1 (en) 2006-01-11 2007-07-19 Raindance Technologies, Inc. Microfluidic devices, methods of use, and kits for performing diagnostics
US9328344B2 (en) 2006-01-11 2016-05-03 Raindance Technologies, Inc. Microfluidic devices and methods of use in the formation and control of nanoreactors
US20180080020A1 (en) 2006-01-11 2018-03-22 Raindance Technologies, Inc. Microfluidic devices and methods of use in the formation and control of nanoreactors
US9752141B2 (en) 2006-01-11 2017-09-05 Raindance Technologies, Inc. Microfluidic devices and methods of use in the formation and control of nanoreactors
EP2363205A2 (en) 2006-01-11 2011-09-07 Raindance Technologies, Inc. Microfluidic Devices And Methods Of Use In The Formation And Control Of Nanoreactors
US20170067047A1 (en) 2006-01-11 2017-03-09 Raindance Technologies, Inc. Microfluidic devices and methods of use in the formation and control of nanoreactors
US20100137163A1 (en) 2006-01-11 2010-06-03 Link Darren R Microfluidic Devices and Methods of Use in The Formation and Control of Nanoreactors
US20070172873A1 (en) 2006-01-23 2007-07-26 Sydney Brenner Molecular counting
US7544473B2 (en) 2006-01-23 2009-06-09 Population Genetics Technologies Ltd. Nucleic acid analysis using sequence tokens
US7537897B2 (en) 2006-01-23 2009-05-26 Population Genetics Technologies, Ltd. Molecular counting
WO2007089541A2 (en) 2006-01-27 2007-08-09 President And Fellows Of Harvard College Fluidic droplet coalescence
US20090075307A1 (en) 2006-01-27 2009-03-19 Tripath Imaging, Inc. Methods for identifying patients with an increased likelihood of having ovarian cancer and compositions therefor
US20070195127A1 (en) 2006-01-27 2007-08-23 President And Fellows Of Harvard College Fluidic droplet coalescence
WO2007092473A2 (en) 2006-02-02 2007-08-16 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive fetal genetic screening by digital analysis
US20070202525A1 (en) 2006-02-02 2007-08-30 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive fetal genetic screening by digital analysis
WO2007092473A3 (en) 2006-02-02 2008-11-13 Univ Leland Stanford Junior Non-invasive fetal genetic screening by digital analysis
US7888017B2 (en) 2006-02-02 2011-02-15 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive fetal genetic screening by digital analysis
US9322511B2 (en) 2006-02-07 2016-04-26 Stokes Bio Limited Liquid bridge and system
US20100216128A1 (en) 2006-02-07 2010-08-26 Stokes Bio Ltd. Methods for analyzing agricultural and environmental samples
US10730051B2 (en) 2006-02-07 2020-08-04 Stokes Bio Ltd. Liquid bridge and system
US20080009005A1 (en) 2006-02-09 2008-01-10 Kruk Patricia A Detection of cancer by elevated levels of BCL-2
US20090215633A1 (en) 2006-03-01 2009-08-27 Keygene N.V. High throughput sequence-based detection of snps using ligation assays
US20070292869A1 (en) 2006-03-02 2007-12-20 Ppd Biomarker Discovery Sciences, Llc Compositions and Methods for Analyzing Renal Cancer
US20080058432A1 (en) 2006-03-03 2008-03-06 Yixin Wang Molecular assay to predict recurrence of Duke's B colon cancer
US20100233083A1 (en) 2006-03-21 2010-09-16 Dsm Ip Assets B.V. Microparticles comprising a crosslinked polymer
US20090127454A1 (en) 2006-03-24 2009-05-21 Phenomenome Discoveries Inc. Biomarkers useful for diagnosing prostate cancer, and methods thereof
WO2007123744A2 (en) 2006-03-31 2007-11-01 Solexa, Inc. Systems and devices for sequence by synthesis analysis
US20100111768A1 (en) 2006-03-31 2010-05-06 Solexa, Inc. Systems and devices for sequence by synthesis analysis
WO2007114794A1 (en) 2006-03-31 2007-10-11 Nam Trung Nguyen Active control for droplet-based microfluidics
US20070241068A1 (en) 2006-04-13 2007-10-18 Pamula Vamsee K Droplet-based washing
US7282337B1 (en) 2006-04-14 2007-10-16 Helicos Biosciences Corporation Methods for increasing accuracy of nucleic acid sequencing
US20070242105A1 (en) 2006-04-18 2007-10-18 Vijay Srinivasan Filler fluids for droplet operations
US20080053205A1 (en) 2006-04-18 2008-03-06 Pollack Michael G Droplet-based particle sorting
US20070275415A1 (en) 2006-04-18 2007-11-29 Vijay Srinivasan Droplet-based affinity assays
US20070243634A1 (en) 2006-04-18 2007-10-18 Pamula Vamsee K Droplet-based surface modification and washing
US20070259351A1 (en) 2006-05-03 2007-11-08 James Chinitz Evaluating Genetic Disorders
US20070259368A1 (en) 2006-05-03 2007-11-08 Genomictree, Inc. Gastric cancer biomarker discovery
EP2047910B1 (en) 2006-05-11 2012-01-11 Raindance Technologies, Inc. Microfluidic device and method
WO2007133710A2 (en) 2006-05-11 2007-11-22 Raindance Technologies, Inc. Microfluidic devices and methods of use thereof
US20170183722A1 (en) 2006-05-11 2017-06-29 Raindance Technologies, Inc. Systems for handling microfluidic droplets
US10639597B2 (en) 2006-05-11 2020-05-05 Bio-Rad Laboratories, Inc. Microfluidic devices
US20080014589A1 (en) 2006-05-11 2008-01-17 Link Darren R Microfluidic devices and methods of use thereof
US20130260447A1 (en) 2006-05-11 2013-10-03 Darren R. Link Systems and methods for handling microfluidic droplets
US9896722B2 (en) 2006-05-11 2018-02-20 Raindance Technologies, Inc. Systems for handling microfluidic droplets
US20180305747A1 (en) 2006-05-11 2018-10-25 Raindance Technologies, Inc. Systems and methods for handling microfluidic droplets
US20180280897A1 (en) 2006-05-11 2018-10-04 Raindance Technologies, Inc. Microfluidic devices
US20080003142A1 (en) 2006-05-11 2008-01-03 Link Darren R Microfluidic devices
US20180272296A1 (en) 2006-05-11 2018-09-27 Raindance Technologies, Inc. Microfluidic devices
US20130210639A1 (en) 2006-05-11 2013-08-15 Darren R. Link Microfluidic devices
US20180272295A1 (en) 2006-05-11 2018-09-27 Raindance Technologies, Inc. Microfluidic devices
US9273308B2 (en) 2006-05-11 2016-03-01 Raindance Technologies, Inc. Selection of compartmentalized screening method
US20130183659A1 (en) 2006-05-11 2013-07-18 Darren R. Link Microfluidic devices
US20170304785A1 (en) 2006-05-11 2017-10-26 Raindance Technologies, Inc. Microfluidic devices
US10625220B2 (en) 2006-05-11 2020-04-21 Bio-Rad Laboratories, Inc. Microfluidic devices
WO2008063227A2 (en) 2006-05-11 2008-05-29 Raindance Technologies, Inc. Microfluidic devices
US10011865B2 (en) 2006-05-11 2018-07-03 Raindance Technologies, Inc. Systems for handling microfluidic droplets
US20200360876A1 (en) 2006-05-11 2020-11-19 Bio-Rad Laboratories, Inc. Microfluidic devices
US20180178174A1 (en) 2006-05-11 2018-06-28 Raindance Technologies, Inc. Microfluidic devices
US9981230B2 (en) 2006-05-11 2018-05-29 Raindance Technologies, Inc. Microfluidic devices
US10927407B2 (en) 2006-05-11 2021-02-23 Bio-Rad Laboratories, Inc. Systems and methods for handling microfluidic droplets
US20180135117A1 (en) 2006-05-11 2018-05-17 Raindance Technologies, Inc. Systems for handling microfluidic droplets
US20210262020A1 (en) 2006-05-11 2021-08-26 Bio-Rad Laboratories, Inc. Systems and methods for handling microfluidic droplets
US9562837B2 (en) 2006-05-11 2017-02-07 Raindance Technologies, Inc. Systems for handling microfludic droplets
US20130090248A1 (en) 2006-05-11 2013-04-11 Darren R. Link Selection of compartmentalised screening
US20100009353A1 (en) 2006-05-18 2010-01-14 Colin Barnes Dye Compounds and the Use of their Labelled Conjugates
US20080124726A1 (en) 2006-05-26 2008-05-29 Althea Technologies, Inc. Biochemical analysis of partitioned cells
US20080081333A1 (en) 2006-05-26 2008-04-03 University Of Maryland, Baltimore Methylated promoters as biomarkers of colon cancer
WO2007140015A2 (en) 2006-05-26 2007-12-06 Althea Technologies, Inc Biochemical analysis of partitioned cells
WO2007138178A2 (en) 2006-05-30 2007-12-06 Centre National De La Recherche Scientifique Method for treating drops in a microfluid circuit
US20120171667A1 (en) 2006-06-14 2012-07-05 Daniel Shoemaker Rare Cell Analysis Using Sample Splitting And DNA Tags
US8715934B2 (en) 2006-06-19 2014-05-06 The Johns Hopkins University Single-molecule PCR on microparticles in water-in-oil emulsions
US9592510B2 (en) 2006-06-30 2017-03-14 Canon U.S. Life Sciences, Inc. Real-time PCR in micro-channels
US20080021330A1 (en) 2006-07-21 2008-01-24 Samsung Electronics Co., Ltd. Apparatus, method and optical sensor module using a tilter for body fat measurement
US20080020940A1 (en) 2006-07-24 2008-01-24 Miraculins Inc. Biomarkers for use in the diagnosis and treatment of colorectal cancer
WO2008021123A1 (en) 2006-08-07 2008-02-21 President And Fellows Of Harvard College Fluorocarbon emulsion stabilizing surfactants
US9498761B2 (en) 2006-08-07 2016-11-22 Raindance Technologies, Inc. Fluorocarbon emulsion stabilizing surfactants
US20150217246A1 (en) 2006-08-07 2015-08-06 Raindance Technologies, Inc. Fluorocarbon emulsion stabilizing surfactants
US9012390B2 (en) 2006-08-07 2015-04-21 Raindance Technologies, Inc. Fluorocarbon emulsion stabilizing surfactants
US20100105112A1 (en) 2006-08-07 2010-04-29 Christian Holtze Fluorocarbon emulsion stabilizing surfactants
US20080050723A1 (en) 2006-08-23 2008-02-28 Nabil Belacel Molecular method for diagnosis of colon cancer
US20080220986A1 (en) 2006-08-24 2008-09-11 Niall Anthony Gormley Method for retaining even coverage of short insert libraries
US20100015617A1 (en) 2006-08-31 2010-01-21 Toyo Seikan Kaisha, Ltd. Nucleic Acid Amplification Method
US20080057514A1 (en) 2006-09-06 2008-03-06 Vanderbilt University Methods of screening for gastrointestinal cancer
US20080063227A1 (en) 2006-09-07 2008-03-13 Kristin Rohrseitz Method for adapting a hearing aid using a genetic feature
US20080081330A1 (en) 2006-09-28 2008-04-03 Helicos Biosciences Corporation Method and devices for analyzing small RNA molecules
US20080216563A1 (en) 2006-09-29 2008-09-11 Reed Wayne F Methods and devices for simultaneously monitoring the characteristics of microscopic particles in suspension and the characteristics of soluble components during reactions
US20080269157A1 (en) 2006-10-10 2008-10-30 The Henry F. Jackson Foundation For Military Medicine Prostate cancer-specific alterations in ERG gene expression and detection and treatment methods based on those alterations
US20080092973A1 (en) 2006-10-20 2008-04-24 Tai-Sol Electronics Co., Ltd. Flexible heat pipe
WO2008052138A2 (en) 2006-10-25 2008-05-02 The Regents Of The University Of California Inline-injection microdevice and microfabricated integrated dna analysis system using same
US20090035770A1 (en) 2006-10-25 2009-02-05 The Regents Of The University Of California Inline-injection microdevice and microfabricated integrated DNA analysis system using same
US20110045462A1 (en) 2006-11-14 2011-02-24 The Regents Of The University Of California Digital analysis of gene expression
US20080176236A1 (en) 2006-11-15 2008-07-24 Ming Sound Tsao Materials and methods for prognosing lung cancer survival
US20080234138A1 (en) 2006-12-08 2008-09-25 Shaughnessy John D TP53 gene expression and uses thereof
US20080138806A1 (en) 2006-12-08 2008-06-12 National Taiwan University Biomarkers and detection methods for gastric diseases
US20080213377A1 (en) 2006-12-08 2008-09-04 Bhatia Sangeeta N Delivery of Nanoparticles and/or Agents to Cells
US20100197507A1 (en) 2006-12-14 2010-08-05 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale fet arrays
US20090127589A1 (en) 2006-12-14 2009-05-21 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20100188073A1 (en) 2006-12-14 2010-07-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale fet arrays
US20100282617A1 (en) 2006-12-14 2010-11-11 Ion Torrent Systems Incorporated Methods and apparatus for detecting molecular interactions using fet arrays
US20090026082A1 (en) 2006-12-14 2009-01-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
JP2008153669A (en) 2006-12-15 2008-07-03 Cree Inc Reflective mounting substrate for light emitting diodes
US20080305482A1 (en) 2006-12-21 2008-12-11 Gen-Probe Incorporated Methods and compositions for nucleic acid amplification
US20080166793A1 (en) 2007-01-04 2008-07-10 The Regents Of The University Of California Sorting, amplification, detection, and identification of nucleic acid subsequences in a complex mixture
US20100055677A1 (en) 2007-01-04 2010-03-04 The Regents Of The University Of California Method for genetic identification of unknown organisms
US20080171078A1 (en) 2007-01-12 2008-07-17 Mark Gray Uniformly sized liposomes
US20080181850A1 (en) 2007-01-29 2008-07-31 Northwestern University Biomarkers For Prostate Cancer
US20090233802A1 (en) 2007-02-02 2009-09-17 Helen Bignell Methods for indexing samples and sequencing multiple polynucleotide templates
US20090075265A1 (en) 2007-02-02 2009-03-19 Orion Genomics Llc Gene methylation in thyroid cancer diagnosis
US20090098543A1 (en) 2007-02-02 2009-04-16 Orion Genomics Llc Gene methylation in lung cancer diagnosis
US20090098542A1 (en) 2007-02-02 2009-04-16 Orion Genomics Llc Gene Methylation in Colon Cancer Diagnosis
WO2008115626A2 (en) 2007-02-05 2008-09-25 Microchip Biotechnologies, Inc. Microfluidic and nanofluidic devices, systems, and applications
US8772046B2 (en) 2007-02-06 2014-07-08 Brandeis University Manipulation of fluids and reactions in microfluidic systems
WO2008097559A2 (en) 2007-02-06 2008-08-14 Brandeis University Manipulation of fluids and reactions in microfluidic systems
US20100136544A1 (en) 2007-03-07 2010-06-03 Jeremy Agresti Assays and other reactions involving droplets
US9029085B2 (en) 2007-03-07 2015-05-12 President And Fellows Of Harvard College Assays and other reactions involving droplets
US9816121B2 (en) 2007-03-07 2017-11-14 President And Fellows Of Harvard College Assays and other reactions involving droplets
US20090081237A1 (en) 2007-03-12 2009-03-26 Dana-Farber Cancer Institute Prognostic, diagnostic, and cancer therapeutic uses of FANCI and FANCI modulating agents
WO2008121342A2 (en) 2007-03-28 2008-10-09 President And Fellows Of Harvard College Emulsions and techniques for formation
US20090012187A1 (en) 2007-03-28 2009-01-08 President And Fellows Of Harvard College Emulsions and Techniques for Formation
US8436993B2 (en) 2007-04-02 2013-05-07 Life Technologies Corporation Methods and systems for controlling the flow of particles for detection
US20090062144A1 (en) 2007-04-03 2009-03-05 Nancy Lan Guo Gene signature for prognosis and diagnosis of lung cancer
US10960397B2 (en) 2007-04-19 2021-03-30 President And Fellows Of Harvard College Manipulation of fluids, fluid components and reactions in microfluidic systems
US20100252118A1 (en) 2007-04-19 2010-10-07 Seth Fraden Manipulation of fluids, fluid components and reactions in microfluidic systems
US10675626B2 (en) 2007-04-19 2020-06-09 President And Fellows Of Harvard College Manipulation of fluids, fluid components and reactions in microfluidic systems
US8592221B2 (en) 2007-04-19 2013-11-26 Brandeis University Manipulation of fluids, fluid components and reactions in microfluidic systems
US20190255530A1 (en) 2007-04-19 2019-08-22 Brandeis University Manipulation of fluids, fluid components and reactions in microfluidic systems
WO2008130623A1 (en) 2007-04-19 2008-10-30 Brandeis University Manipulation of fluids, fluid components and reactions in microfluidic systems
US10357772B2 (en) 2007-04-19 2019-07-23 President And Fellows Of Harvard College Manipulation of fluids, fluid components and reactions in microfluidic systems
US20080261295A1 (en) 2007-04-20 2008-10-23 William Frank Butler Cell Sorting System and Methods
US20100130369A1 (en) 2007-04-23 2010-05-27 Advanced Liquid Logic, Inc. Bead-Based Multiplexed Analytical Methods and Instrumentation
WO2008134153A1 (en) 2007-04-23 2008-11-06 Advanced Liquid Logic, Inc. Bead-based multiplexed analytical methods and instrumentation
US20080274908A1 (en) 2007-05-04 2008-11-06 Dermtech International Diagnosis of melanoma by nucleic acid analysis
US20080280302A1 (en) 2007-05-09 2008-11-13 The Regents Of The University Of California Multigene diagnostic assay for malignant thyroid neoplasm
US20090029372A1 (en) 2007-05-14 2009-01-29 Kobenhavns Universitet Adam12 as a biomarker for bladder cancer
US20090105959A1 (en) 2007-06-01 2009-04-23 Braverman Michael S System and method for identification of individual samples from a multiplex mixture
US20100183504A1 (en) 2007-06-14 2010-07-22 Fanqing Frank Chen Multimodal imaging probes for in vivo targeted and non-targeted imaging and therapeutics
US20080311570A1 (en) 2007-06-15 2008-12-18 National Defense Medical Center Cancer screening method
US20090021728A1 (en) 2007-06-21 2009-01-22 Gen-Probe Incorporated Multi-Channel Optical Measurement Instrument
US20090004687A1 (en) 2007-06-29 2009-01-01 Mansfield Brian C Predictive markers for ovarian cancer
US20090017463A1 (en) 2007-07-10 2009-01-15 Vanderbilt University Methods for predicting prostate cancer recurrence
US20120015382A1 (en) 2007-07-13 2012-01-19 President And Fellows Of Harvard College Droplet-based selection
US20090068170A1 (en) 2007-07-13 2009-03-12 President And Fellows Of Harvard College Droplet-based selection
US20090053732A1 (en) 2007-07-16 2009-02-26 Ophir Vermesh Microfluidic devices, methods and systems for detecting target molecules
EP2017910A1 (en) 2007-07-20 2009-01-21 BIOTRONIK CRM Patent AG Active element and battery and method for production of same
US20100285975A1 (en) 2007-07-24 2010-11-11 The Regents Of The University Of California Microfabricated droplet generator for single molecule/cell genetic analysis in engineered monodispersed emulsions
WO2009015296A1 (en) 2007-07-24 2009-01-29 The Regents Of The University Of California Microfabricated dropley generator
US20090042737A1 (en) 2007-08-09 2009-02-12 Katz Andrew S Methods and Devices for Correlated, Multi-Parameter Single Cell Measurements and Recovery of Remnant Biological Material
WO2009029229A2 (en) 2007-08-24 2009-03-05 President And Fellows Of Harvard College Ferrofluid emulsions, particles, and systems and methods for making and using the same
US20090087849A1 (en) 2007-09-06 2009-04-02 Tripath Imaging, Inc. Nucleic acid-based methods and compositions for the detection of ovarian cancer
US20090069194A1 (en) 2007-09-07 2009-03-12 Fluidigm Corporation Copy number variation determination, methods and systems
US20100304378A1 (en) 2007-09-17 2010-12-02 Universite De Strasbourg Method for Detecting or Quantifying a Truncating Mutation
WO2009037266A2 (en) 2007-09-17 2009-03-26 Universite Louis Pasteur Method for detecting or quantifying a truncating mutation
US8318434B2 (en) 2007-09-21 2012-11-27 Katholieke Universiteit Leuven, K.U.Leuven R&D Method for introducing a sample specific DNA tag into a plurality of DNA fragments from a plurality of samples
US20090098555A1 (en) 2007-09-26 2009-04-16 President And Fellows Of Harvard College Methods and applications for stitched dna barcodes
US20090124569A1 (en) 2007-10-12 2009-05-14 Northwestern University Inhibition and treatment of prostate cancer metastasis
WO2009049889A1 (en) 2007-10-16 2009-04-23 Roche Diagnostics Gmbh High resolution, high throughput hla genotyping by clonal sequencing
US20090169482A1 (en) 2007-10-16 2009-07-02 Shiying Zheng Silica-cored carrier particle
US20110053151A1 (en) 2007-11-07 2011-03-03 The University Of British Columbia Microfluidic device and method of using same
WO2009059430A1 (en) 2007-11-07 2009-05-14 The University Of British Columbia Microfluidic device and method of using same
US20090124789A1 (en) 2007-11-09 2009-05-14 Yoshida Eri Polymer microparticles and production method for the same
US8462269B2 (en) 2007-11-16 2013-06-11 Mediatek Inc. Devices and methods for extracting a synchronization signal from a video signal
US20110033854A1 (en) 2007-12-05 2011-02-10 Complete Genomics, Inc. Methods and compositions for long fragment read sequencing
WO2009085929A1 (en) 2007-12-20 2009-07-09 The Polymer Technology Group, Inc. Hybrid polyurethane block copolymers with thermoplastic processability and thermoset properties
US20110267457A1 (en) 2007-12-21 2011-11-03 David A Weitz Systems and methods for nucleic acid sequencing
US20100273173A1 (en) 2007-12-26 2010-10-28 Arkray, Inc. Method for amplifying target nucleic acid sequence and probe used for the same
US20090226971A1 (en) 2008-01-22 2009-09-10 Neil Reginald Beer Portable Rapid Microfluidic Thermal Cycler for Extremely Fast Nucleic Acid Amplification
US20090226972A1 (en) 2008-01-22 2009-09-10 Neil Reginald Beer Rapid Microfluidic Thermal Cycler for Nucleic Acid Amplification
WO2009094623A2 (en) 2008-01-24 2009-07-30 Balter, Inc. Method for discriminating between malignant and benign tissue lesions
WO2009117485A2 (en) 2008-03-18 2009-09-24 Balter, Inc. Optical method for determining morphological parameters and physiological properties of tissue
US20090246788A1 (en) 2008-04-01 2009-10-01 Roche Nimblegen, Inc. Methods and Assays for Capture of Nucleic Acids
JP2009265751A (en) 2008-04-22 2009-11-12 Oki Electric Ind Co Ltd Character recognition device, optical character recognition system and character recognition program
US20110190146A1 (en) 2008-04-28 2011-08-04 President And Fellows Of Harvard College Microfluidic device for storage and well-defined arrangement of droplets
US20110104725A1 (en) 2008-05-02 2011-05-05 Advanced Liquid Logic, Inc. Method of Effecting Coagulation in a Droplet
US20110104816A1 (en) 2008-05-03 2011-05-05 Advanced Liquid Logic, Inc. Method of Loading a Droplet Actuator
WO2009137415A2 (en) 2008-05-03 2009-11-12 Advanced Liquid Logic, Inc. Reagent and sample preparation, loading, and storage
US20100075436A1 (en) 2008-05-06 2010-03-25 Urdea Michael S Methods for use with nanoreactors
US20100021984A1 (en) 2008-05-23 2010-01-28 Edd Jon F Microfluidic Droplet Encapsulation
US20100124759A1 (en) 2008-06-27 2010-05-20 Massachusetts Institute Of Technology Microfluidic droplets for metabolic engineering and other applications
US20090325217A1 (en) 2008-06-30 2009-12-31 Microbix Biosystems Inc. Method and apparatus for sorting cells
US20100003687A1 (en) 2008-07-01 2010-01-07 454 Life Sciences Corporation System and method for detection of HIV tropism variants
US20110275063A1 (en) 2008-07-11 2011-11-10 President And Fellows Of Harvard College Systems and methods of droplet-based selection
US20110188717A1 (en) 2008-07-15 2011-08-04 Universite Pierre Et Marie Curie (Paris 6) Method and device for reading an emulsion
US10533998B2 (en) 2008-07-18 2020-01-14 Bio-Rad Laboratories, Inc. Enzyme quantification
WO2010009365A1 (en) 2008-07-18 2010-01-21 Raindance Technologies, Inc. Droplet libraries
US20200225232A1 (en) 2008-07-18 2020-07-16 Bio-Rad Laboratories, Inc. Enzyme quantification
US20120264646A1 (en) 2008-07-18 2012-10-18 Raindance Technologies, Inc. Enzyme quantification
US20100022414A1 (en) 2008-07-18 2010-01-28 Raindance Technologies, Inc. Droplet Libraries
US20220047998A1 (en) 2008-07-18 2022-02-17 Bio-Rad Laboratories, Inc. Droplet libraries
US20220050108A1 (en) 2008-07-18 2022-02-17 Bio-Rad Laboratories, Inc. Enzyme quantification
US20170028365A1 (en) 2008-07-18 2017-02-02 Raindance Technologies, Inc. Droplet Libraries
US20180353913A1 (en) 2008-07-18 2018-12-13 Raindance Technologies, Inc. Droplet libraries
US20220042995A1 (en) 2008-07-18 2022-02-10 Bio-Rad Laboratories, Inc. Enzyme quantification
US20100035252A1 (en) 2008-08-08 2010-02-11 Ion Torrent Systems Incorporated Methods for sequencing individual nucleic acids under tension
US10626451B2 (en) 2008-08-12 2020-04-21 Stokes Bio Limited Methods and devices for digital PCR
US20110218123A1 (en) 2008-09-19 2011-09-08 President And Fellows Of Harvard College Creation of libraries of droplets and related species
US20150336072A1 (en) 2008-09-19 2015-11-26 President And Fellows Of Harvard College Creation of libraries of droplets and related species
US20100173394A1 (en) 2008-09-23 2010-07-08 Colston Jr Billy Wayne Droplet-based assay system
US20110311978A1 (en) 2008-09-23 2011-12-22 Quantalife, Inc. System for detection of spaced droplets
WO2010040006A1 (en) 2008-10-02 2010-04-08 Blomberg Jerome O Curbless multiple skylight system and smoke vent system
US20100086914A1 (en) 2008-10-03 2010-04-08 Roche Molecular Systems, Inc. High resolution, high throughput hla genotyping by clonal sequencing
US20100300559A1 (en) 2008-10-22 2010-12-02 Ion Torrent Systems, Inc. Fluidics system for sequential delivery of reagents
US20100137143A1 (en) 2008-10-22 2010-06-03 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
US20100120098A1 (en) 2008-10-24 2010-05-13 Epicentre Technologies Corporation Transposon end compositions and methods for modifying nucleic acids
WO2010056728A1 (en) 2008-11-11 2010-05-20 Helicos Biosciences Corporation Nucleic acid encoding for multiplex analysis
US9080056B2 (en) 2008-11-26 2015-07-14 University College Cork—National University of Ireland, Cork Process for preparing silica microparticles
US20110111981A1 (en) 2008-12-04 2011-05-12 Masschusetts Institute Of Technology Method for detecting active and latent virally infected cells
US20120015822A1 (en) 2008-12-19 2012-01-19 President And Fellows Of Harvard College Particle-assisted nucleic acid sequencing
US20100184069A1 (en) 2009-01-21 2010-07-22 Streck, Inc. Preservation of fetal nucleic acids in maternal plasma
US20100248237A1 (en) 2009-02-25 2010-09-30 Thomas Froehlich Miniaturized, high-throughput nucleic acid analysis
JP2010198393A (en) 2009-02-26 2010-09-09 Alpine Electronics Inc Map display device
JP2010217477A (en) 2009-03-17 2010-09-30 Yamaha Corp Waveform data generating method
US20100240101A1 (en) 2009-03-19 2010-09-23 Massachusetts Institute Of Technology Parallel Proximity Ligation Event Analysis
US20120231972A1 (en) 2009-03-19 2012-09-13 Golyshin Peter N Probe Compound for Detecting and Isolating Enzymes and Means and Methods Using the Same
US20170336306A1 (en) 2009-03-23 2017-11-23 Raindance Technologies, Inc. Manipulation of microfluidic droplets
US20110000560A1 (en) 2009-03-23 2011-01-06 Raindance Technologies, Inc. Manipulation of Microfluidic Droplets
US9341594B2 (en) 2009-03-23 2016-05-17 Raindance Technologies, Inc. Manipulation of microfluidic droplets
US9733168B2 (en) 2009-03-23 2017-08-15 Raindance Technologies, Inc. Manipulation of microfluidic droplets
US20160209303A1 (en) 2009-03-23 2016-07-21 Raindance Technologies, Inc. Manipulation of microfluidic droplets
US8528589B2 (en) 2009-03-23 2013-09-10 Raindance Technologies, Inc. Manipulation of microfluidic droplets
US20140076430A1 (en) 2009-03-23 2014-03-20 Raindance Technologies, Inc. Manipulation of microfluidic droplets
US10527529B2 (en) 2009-03-23 2020-01-07 Bio-Rad Laboratories, Inc. Manipulation of microfluidic droplets
US20150027892A1 (en) 2009-03-23 2015-01-29 Raindance Technologies, Inc. Manipulation of microfluidic droplets
US20200225129A1 (en) 2009-03-23 2020-07-16 Bio-Rad Laboratories, Inc. Manipulation of microfluidic droplets
US8857462B2 (en) 2009-03-23 2014-10-14 Raindance Technologies, Inc. Manipulation of microfluidic droplets
WO2010115154A1 (en) 2009-04-02 2010-10-07 Fluidigm Corporation Multi-primer amplification method for barcoding of target nucleic acids
US20120252012A1 (en) 2009-05-14 2012-10-04 Assistance Publique - Hopitaux De Marseille Method for detecting prokaryotic dna from a feces sample
US20100304982A1 (en) 2009-05-29 2010-12-02 Ion Torrent Systems, Inc. Scaffolded nucleic acid polymer particles and methods of making and using
US20100301398A1 (en) 2009-05-29 2010-12-02 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
US20100300895A1 (en) 2009-05-29 2010-12-02 Ion Torrent Systems, Inc. Apparatus and methods for performing electrochemical reactions
WO2010151776A2 (en) 2009-06-26 2010-12-29 President And Fellows Of Harvard College Fluid injection
US20120132288A1 (en) 2009-06-26 2012-05-31 President And Fellows Of Harvard College Fluid injection
US8673595B2 (en) 2009-06-29 2014-03-18 Kabushiki Kaisha Toshiba Sample analysis method and assay kit used therein
WO2011005221A1 (en) 2009-07-07 2011-01-13 Agency For Science, Technology And Research Methods of identifying a pair of binding partners
US20130143749A1 (en) 2009-07-07 2013-06-06 John Farid Ghadessy Methods of identifying a pair of binding partners
US20130288254A1 (en) 2009-08-13 2013-10-31 Advanced Liquid Logic, Inc. Droplet Actuator and Droplet-Based Techniques
US20120165219A1 (en) 2009-09-01 2012-06-28 Koninklijke Philips Electronics N.V. Devices and methods for microarray selection
US20110053798A1 (en) 2009-09-02 2011-03-03 Quantalife, Inc. System for mixing fluids by coalescence of multiple emulsions
US20110059556A1 (en) 2009-09-04 2011-03-10 The Research Foundation Of State University Of New York Rapid and Continuous Analyte Processing in Droplet Microfluidic Devices
US10520500B2 (en) 2009-10-09 2019-12-31 Abdeslam El Harrak Labelled silica-based nanomaterial with enhanced properties and uses thereof
WO2011042564A1 (en) 2009-10-09 2011-04-14 Universite De Strasbourg Labelled silica-based nanomaterial with enhanced properties and uses thereof
US20130064776A1 (en) 2009-10-09 2013-03-14 Universite De Strasbourg Labelled silica-based nanomaterial with enhanced properties and uses thereof
US20200217843A1 (en) 2009-10-09 2020-07-09 Abdeslam El Harrak Labelled silica-based nanomaterial with enhanced properties and uses thereof
US20110159499A1 (en) 2009-11-25 2011-06-30 Quantalife, Inc. Methods and compositions for detecting genetic material
US20110151444A1 (en) 2009-12-18 2011-06-23 Andreas Albers Method for detection of an rna molecule, a kit and use related therefor
US10837883B2 (en) 2009-12-23 2020-11-17 Bio-Rad Laboratories, Inc. Microfluidic systems and methods for reducing the exchange of molecules between droplets
US20210088424A1 (en) 2009-12-23 2021-03-25 Bio-Rad Laboratories, Inc. Microfluidic systems and methods for reducing the exchange of molecules between droplets
US20130109575A1 (en) 2009-12-23 2013-05-02 Raindance Technologies, Inc. Microfluidic systems and methods for reducing the exchange of molecules between droplets
WO2011079176A2 (en) 2009-12-23 2011-06-30 Raindance Technologies, Inc. Microfluidic systems and methods for reducing the exchange of molecules between droplets
US20120010085A1 (en) 2010-01-19 2012-01-12 Rava Richard P Methods for determining fraction of fetal nucleic acids in maternal samples
US20190330683A1 (en) 2010-02-12 2019-10-31 Bio-Rad Laboratories, Inc. Digital analyte analysis
US9366632B2 (en) 2010-02-12 2016-06-14 Raindance Technologies, Inc. Digital analyte analysis
US20130224751A1 (en) 2010-02-12 2013-08-29 Jeffrey Olson Digital analyte analysis
US20180057863A1 (en) 2010-02-12 2018-03-01 Raindance Technologies, Inc. Digital analyte analysis
WO2011100604A2 (en) 2010-02-12 2011-08-18 Raindance Technologies, Inc. Digital analyte analysis
US9074242B2 (en) 2010-02-12 2015-07-07 Raindance Technologies, Inc. Digital analyte analysis
EP2534267B1 (en) 2010-02-12 2018-04-11 Raindance Technologies, Inc. Digital analyte analysis
US20120302448A1 (en) 2010-02-12 2012-11-29 Raindance Technologies, Inc. Digital analyte analysis
US20110244455A1 (en) 2010-02-12 2011-10-06 Raindance Technologies, Inc. Digital analyte analysis
US20110250597A1 (en) 2010-02-12 2011-10-13 Raindance Technologies, Inc. Digital analyte analysis
US20110257031A1 (en) 2010-02-12 2011-10-20 Life Technologies Corporation Nucleic acid, biomolecule and polymer identifier codes
US9127310B2 (en) 2010-02-12 2015-09-08 Raindance Technologies, Inc. Digital analyte analysis
US10612081B2 (en) 2010-02-12 2020-04-07 Bio-Rad Laboratories, Inc. Digital analyte analysis
US9745617B2 (en) 2010-02-12 2017-08-29 Raindance Technologies, Inc. Digital analyte analysis
US20200318157A1 (en) 2010-02-12 2020-10-08 Bio-Rad Laboratories, Inc. Digital analyte analysis
US10808279B2 (en) 2010-02-12 2020-10-20 Bio-Rad Laboratories, Inc. Digital analyte analysis
US20140329239A1 (en) 2010-02-12 2014-11-06 Raindance Technologies, Inc. Digital analyte analysis
US20130295567A1 (en) 2010-02-12 2013-11-07 Darren Roy Link Digital analyte analysis
US20140045712A1 (en) 2010-02-12 2014-02-13 Darren Roy Link Digital analyte analysis
US8535889B2 (en) 2010-02-12 2013-09-17 Raindance Technologies, Inc. Digital analyte analysis
US20170002400A1 (en) 2010-02-12 2017-01-05 Raindance Technologies, Inc. Digital analyte analysis
US9228229B2 (en) 2010-02-12 2016-01-05 Raindance Technologies, Inc. Digital analyte analysis
US20130295568A1 (en) 2010-02-12 2013-11-07 Darren Roy Link Digital analyte analysis
US10351905B2 (en) 2010-02-12 2019-07-16 Bio-Rad Laboratories, Inc. Digital analyte analysis
US9494520B2 (en) 2010-02-12 2016-11-15 Raindance Technologies, Inc. Digital analyte analysis
US9441266B2 (en) 2010-02-12 2016-09-13 Raindance Technologies, Inc. Digital analyte analysis
US20160222433A1 (en) 2010-02-12 2016-08-04 Raindance Technologies, Inc. Digital analyte analysis
US20130196324A1 (en) 2010-02-12 2013-08-01 Jonathan William Larson Digital analyte analysis
US9399797B2 (en) 2010-02-12 2016-07-26 Raindance Technologies, Inc. Digital analyte analysis
US20210002703A1 (en) 2010-02-12 2021-01-07 Bio-Rad Laboratories, Inc. Digital analyte analysis
US20120014977A1 (en) 2010-02-19 2012-01-19 National University Corporation Chiba University NOVEL ALTERNATIVE SPLICING VARIANT OF OATP1B3 mRNA
US20130203606A1 (en) 2010-02-25 2013-08-08 Advanced Liquid Logic Inc Method of Preparing a Nucleic Acid Library
US20110223314A1 (en) 2010-03-10 2011-09-15 Xiaoxiao Zhang Efficient microencapsulation
US20120190032A1 (en) 2010-03-25 2012-07-26 Ness Kevin D Droplet generation for droplet-based assays
US20110274706A1 (en) 2010-05-04 2011-11-10 General Electric Company Nucleic acid delivery vehicle and uses thereof
US20130123339A1 (en) 2010-05-12 2013-05-16 Protiva Biotherapeutics Inc Compositions and methods for silencing apolipoprotein b
US20120021919A1 (en) 2010-07-23 2012-01-26 Thomas Scholl Identification of Differentially Represented Fetal or Maternal Genomic Regions and Uses Thereof
WO2012022976A1 (en) 2010-08-20 2012-02-23 Sphere Fluidics Limited Method of providing a chemical or biological material in quantised form and system therefor
WO2012036679A1 (en) 2010-09-15 2012-03-22 Life Technologies Corporation Methods and apparatus for measuring analytes
US20140235452A1 (en) 2010-09-15 2014-08-21 Life Technologies Corporation Methods and apparatus for measuring analytes
US9562897B2 (en) 2010-09-30 2017-02-07 Raindance Technologies, Inc. Sandwich assays in droplets
US20120122714A1 (en) 2010-09-30 2012-05-17 Raindance Technologies, Inc. Sandwich assays in droplets
US10761090B2 (en) 2010-09-30 2020-09-01 Bio-Rad Laboratories, Inc. Sandwich assays in droplets
US20170176429A1 (en) 2010-09-30 2017-06-22 Raindance Technologies, Inc. Sandwich assays in droplets
US20210041432A1 (en) 2010-09-30 2021-02-11 Bio-Rad Laboratories, Inc. Sandwich assays in droplets
WO2012045012A2 (en) 2010-09-30 2012-04-05 Raindance Technologies, Inc. Sandwich assays in droplets
US20130296535A1 (en) 2010-10-08 2013-11-07 President And Fellows Of Harvard College High-Throughput Immune Sequencing
WO2012047297A2 (en) 2010-10-08 2012-04-12 Chen Gao Highly multiplexed real-time pcr using encoded microbeads
US20120088691A1 (en) 2010-10-08 2012-04-12 Gao Chen Highly multiplexed real-time pcr using encoded microbeads
WO2012048341A1 (en) 2010-10-08 2012-04-12 President And Fellows Of Harvard College High-throughput single cell barcoding
US20130274117A1 (en) 2010-10-08 2013-10-17 President And Fellows Of Harvard College High-Throughput Single Cell Barcoding
US20130225623A1 (en) 2010-10-27 2013-08-29 Mount Sinai School Of Medicine Methods of Treating Psychiatric or Neurological Disorders with MGLUR Antagonists
US8278711B2 (en) 2010-11-23 2012-10-02 General Electric Company Semiconductor device and method of making the same
WO2012083225A2 (en) 2010-12-16 2012-06-21 Gigagen, Inc. System and methods for massively parallel analysis of nycleic acids in single cells
US20140057799A1 (en) 2010-12-16 2014-02-27 Gigagen System and Methods for Massively Parallel Analysis of Nucleic Acids in Single Cells
US20120167142A1 (en) 2010-12-23 2012-06-28 Eldon Technology Limited Methods and apparatuses to facilitate preselection of programming preferences
US20120164652A1 (en) 2010-12-27 2012-06-28 Ibis Biosciences, Inc. Quantitating high titer samples by digital pcr
WO2012103339A1 (en) 2011-01-26 2012-08-02 Firestone Industrial Products Company, Llc Gas spring piston as well as gas spring assembly, suspension system, kit and method including same
US20120244043A1 (en) 2011-01-28 2012-09-27 Sean Leblanc Elastomeric gasket for fluid interface to a microfluidic chip
WO2012106385A2 (en) 2011-01-31 2012-08-09 Apprise Bio, Inc. Methods of identifying multiple epitopes in cells
US10144950B2 (en) 2011-01-31 2018-12-04 Roche Sequencing Solutions, Inc. Methods of identifying multiple epitopes in cells
US20120288857A1 (en) 2011-02-03 2012-11-15 Fluidigm Corporation Multifunctional probe-primers
US20120322058A1 (en) 2011-02-09 2012-12-20 Bio-Rad Laboratories Analysis of nucleic acids
US20120208705A1 (en) 2011-02-10 2012-08-16 Steemers Frank J Linking sequence reads using paired code tags
US10155207B2 (en) 2011-02-11 2018-12-18 Bio-Rad Laboratories, Inc. Methods for forming mixed droplets
US20120208241A1 (en) 2011-02-11 2012-08-16 Raindance Technologies, Inc. Thermocycling device for nucleic acid amplification and methods of use
US11077415B2 (en) 2011-02-11 2021-08-03 Bio-Rad Laboratories, Inc. Methods for forming mixed droplets
US9364803B2 (en) 2011-02-11 2016-06-14 Raindance Technologies, Inc. Methods for forming mixed droplets
US20120219947A1 (en) 2011-02-11 2012-08-30 Raindance Technologies, Inc. Methods for forming mixed droplets
US20160346748A1 (en) 2011-02-11 2016-12-01 Raindance Technologies, Inc. Methods for forming mixed droplets
US9266104B2 (en) 2011-02-11 2016-02-23 Raindance Technologies, Inc. Thermocycling device for nucleic acid amplification and methods of use
US20190134581A1 (en) 2011-02-11 2019-05-09 Bio-Rad Laboratories, Inc. Methods for forming mixed droplets
US20190316119A1 (en) 2011-02-18 2019-10-17 Raindance Technologies, Inc. Compositions and methods for molecular labeling
US20200399635A1 (en) 2011-02-18 2020-12-24 Bio-Rad Laboratories, Inc. Compositions and methods for molecular labeling
US20160201125A1 (en) 2011-02-18 2016-07-14 Raindance Technologies, Inc. Compositions and methods for molecular labeling
US9150852B2 (en) 2011-02-18 2015-10-06 Raindance Technologies, Inc. Compositions and methods for molecular labeling
US11168353B2 (en) 2011-02-18 2021-11-09 Bio-Rad Laboratories, Inc. Compositions and methods for molecular labeling
US10584332B2 (en) 2011-02-18 2020-03-10 Bio-Rad Laboratories, Inc. Compositions and methods for molecular labeling
US20200270600A1 (en) 2011-02-18 2020-08-27 Bio-Rad Laboratories, Inc. Compositions and methods for molecular labeling
US20160289670A1 (en) 2011-02-18 2016-10-06 Raindance Technologies, Inc. Compositions and methods for molecular labeling
US20120220494A1 (en) 2011-02-18 2012-08-30 Raindance Technolgies, Inc. Compositions and methods for molecular labeling
US20140155274A1 (en) 2011-03-24 2014-06-05 President And Fellows Of Harvard College Single Cell Nucleic Acid Detection and Analysis
JP2012204765A (en) 2011-03-28 2012-10-22 Tamura Seisakusho Co Ltd Reflow device
US20120258516A1 (en) 2011-04-07 2012-10-11 Life Technologies Corporation System and Methods for Making and Processing Emulsions
WO2012142213A2 (en) 2011-04-15 2012-10-18 The Johns Hopkins University Safe sequencing system
US20130244906A1 (en) 2011-05-05 2013-09-19 Biopico Systems Microfluidic devices and methods based on massively parallel picoreactors for cell and molecular diagnostics
US20200249230A1 (en) 2011-06-02 2020-08-06 Bio-Rad Laboratories, Inc. Enzyme quantification
WO2012167142A2 (en) 2011-06-02 2012-12-06 Raindance Technolgies, Inc. Enzyme quantification
US20120309002A1 (en) 2011-06-02 2012-12-06 Raindance Technologies, Inc. Sample multiplexing
US9556470B2 (en) 2011-06-02 2017-01-31 Raindance Technologies, Inc. Enzyme quantification
US10551382B2 (en) 2011-06-02 2020-02-04 Bio-Rad Laboratories, Inc. Enzyme quantification
US20140295421A1 (en) 2011-06-02 2014-10-02 Raindance Technologies, Inc. Enzyme quantification
US8841071B2 (en) 2011-06-02 2014-09-23 Raindance Technologies, Inc. Sample multiplexing
US10139411B2 (en) 2011-06-02 2018-11-27 Raindance Technologies, Inc. Enzyme quantification
US20140256568A1 (en) 2011-06-02 2014-09-11 Raindance Technologies, Inc. Sample multiplexing
US20170131279A1 (en) 2011-06-02 2017-05-11 Raindance Technologies, Inc. Enzyme quantification
US20190094226A1 (en) 2011-06-02 2019-03-28 Raindance Technologies, Inc. Enzyme quantification
US20130178378A1 (en) 2011-06-09 2013-07-11 Andrew C. Hatch Multiplex digital pcr
US20130143745A1 (en) 2011-06-24 2013-06-06 QB3/Pharm-Chem Digital Health Garage Compositions and methods for identifying the essential genome of an organism
US20130099018A1 (en) 2011-07-20 2013-04-25 Raindance Technolgies, Inc. Manipulating droplet size
US20160281140A1 (en) 2011-07-20 2016-09-29 Raindance Technologies, Inc. Manipulating droplet size
US8658430B2 (en) 2011-07-20 2014-02-25 Raindance Technologies, Inc. Manipulating droplet size
US20140154695A1 (en) 2011-07-20 2014-06-05 Raindance Technologies, Inc. Manipulating droplet size
US20200002748A1 (en) 2011-07-20 2020-01-02 Bio-Rad Laboratories, Inc. Manipulating droplet size
US10428369B2 (en) 2011-07-20 2019-10-01 Bio-Rad Laboratories, Inc. Analyzing microdroplet outline size and adjusting channel pressure to alter microdroplet size
WO2013014356A2 (en) 2011-07-28 2013-01-31 Valeo Equipements Electriques Moteur Motor vehicle starter circuit comprising a voltage raising device and starter so equipped
US20130157870A1 (en) 2011-09-09 2013-06-20 The Board Of Trustees Of The Leland Stanford Junior University Methods for obtaining a sequence
US20130109577A1 (en) 2011-10-14 2013-05-02 Pacific Biosciences Of California, Inc. Real-time redox sequencing
US20130217071A1 (en) 2011-12-30 2013-08-22 Luz Montesclaros Methods and compositions for performing nucleic acid amplification reactions
US20150018236A1 (en) 2012-02-02 2015-01-15 Invenra Inc. High throughput screen for biologically active polypeptides
US20130210638A1 (en) 2012-02-10 2013-08-15 Jeffrey Charles Olson Methods for sequencing nucleic acid
US20150126400A1 (en) 2012-02-10 2015-05-07 Raindance Technologies, Inc. Molecular diagnostic screening assay
WO2013120089A1 (en) 2012-02-10 2013-08-15 Raindance Technologies, Inc. Molecular diagnostic screening assay
US20130210659A1 (en) 2012-02-10 2013-08-15 Andrew Watson Molecular diagnostic screening assay
US20130225418A1 (en) 2012-02-24 2013-08-29 Andrew Watson Labeling and sample preparation for sequencing
US9176031B2 (en) 2012-02-24 2015-11-03 Raindance Technologies, Inc. Labeling and sample preparation for sequencing
WO2013165748A1 (en) 2012-04-30 2013-11-07 Raindance Technologies, Inc Digital analyte analysis
WO2014026031A1 (en) 2012-08-10 2014-02-13 Sequenta, Inc. High sensitivity mutation detection using sequence tags
US20140065631A1 (en) * 2012-08-31 2014-03-06 Roche Molecular Systems, Inc. Microfluidic Chip, Device and System for the Generation of Aqueous Droplets in Emulsion Oil for Nucleic Acid Amplification
US20150247191A1 (en) 2012-09-12 2015-09-03 The Regents Of The University Of California Accurate genome sequencing of single cells by single-stranded amplification and sequencing
CN104812915A (en) 2012-10-22 2015-07-29 新加坡国立大学 Assay for the parallel detection of biological material based on PCR
US20180100185A1 (en) 2012-10-22 2018-04-12 Raindance Technologies, Inc. Methods for analyzing dna
US20140113300A1 (en) 2012-10-22 2014-04-24 Raindance Technologies, Inc. Methods for analyzing dna
US9840734B2 (en) 2012-10-22 2017-12-12 Raindance Technologies, Inc. Methods for analyzing DNA
US10724082B2 (en) 2012-10-22 2020-07-28 Bio-Rad Laboratories, Inc. Methods for analyzing DNA
WO2014065756A1 (en) 2012-10-22 2014-05-01 National University Of Singapore Assay for the parallel detection of biological material based on pcr
US20150258520A1 (en) 2012-11-30 2015-09-17 The Broad Institute Inc. High-throughput dynamic reagent delivery system
US20140256585A1 (en) 2013-03-07 2014-09-11 Bio-Rad Laboratories, Inc. Repetitive reverse transcription partition assay
US9273349B2 (en) 2013-03-14 2016-03-01 Affymetrix, Inc. Detection of nucleic acids
US20140274786A1 (en) 2013-03-15 2014-09-18 Bio-Rad Laboratories, Inc. Digital assays with associated targets
WO2014165559A2 (en) 2013-04-02 2014-10-09 Raindance Technologies, Inc. Systems and methods for handling microfluidic droplets
WO2014194131A2 (en) 2013-05-29 2014-12-04 Gnubio, Inc. Systems and methods for sequencing in emulsion based microfluidics
WO2014204939A2 (en) 2013-06-17 2014-12-24 Kim Lewis Methods for quantitative determination of protein-nucleic acid interactions in complex mixtures
US20150038356A1 (en) 2013-07-25 2015-02-05 Bio-Rad Laboratories, Inc. Genetic assays
WO2015013681A1 (en) 2013-07-25 2015-01-29 Bio-Rad Laboratories, Inc. Genetic assays
US20150099266A1 (en) 2013-10-04 2015-04-09 Raindance Technologies, Inc. Digital analysis of nucleic acid modification
US20160304954A1 (en) 2013-12-11 2016-10-20 Accuragen, Inc. Compositions and methods for detecting rare sequence variants
US20180223348A1 (en) 2013-12-12 2018-08-09 Raindance Technologies, Inc. Distinguishing rare variations in a nucleic acid sequence from a sample
US11174509B2 (en) 2013-12-12 2021-11-16 Bio-Rad Laboratories, Inc. Distinguishing rare variations in a nucleic acid sequence from a sample
US20150167066A1 (en) 2013-12-12 2015-06-18 Raindance Technologies, Inc. Distinguishing rare variations in a nucleic acid sequence from a sample
US9944977B2 (en) 2013-12-12 2018-04-17 Raindance Technologies, Inc. Distinguishing rare variations in a nucleic acid sequence from a sample
US20150184256A1 (en) 2013-12-31 2015-07-02 Raindance Technologies, Inc. System and method for detection of rna species
US20150197790A1 (en) 2014-01-10 2015-07-16 Bio-Rad Laboratories, Inc. Intercalating dyes for differential detection
WO2015164212A1 (en) 2014-04-21 2015-10-29 President And Fellows Of Harvard College Systems and methods for barcoding nucleic acids
US10596541B2 (en) 2014-04-21 2020-03-24 President And Fellows Of Harvard College Systems and methods for barcoding nucleic acids
US20180304222A1 (en) 2014-04-21 2018-10-25 President And Fellows Of Harvard College Systems and methods for barcoding nucleic acids
WO2015200541A1 (en) 2014-06-24 2015-12-30 Bio-Rad Laboratories, Inc. Digital pcr barcoding
US20160060621A1 (en) 2014-06-24 2016-03-03 Bio-Rad Laboratories, Inc. Digital pcr barcoding
WO2015200893A2 (en) 2014-06-26 2015-12-30 10X Genomics, Inc. Methods of analyzing nucleic acids from individual cells or cell populations
US20160200847A1 (en) 2014-12-12 2016-07-14 Marcella Chiari Clickable polymers and gels for microarray and other applications
US9857202B2 (en) 2015-05-28 2018-01-02 Sanyo Denki Co., Ltd. Sensor for motor
US10647981B1 (en) 2015-09-08 2020-05-12 Bio-Rad Laboratories, Inc. Nucleic acid library generation methods and compositions
US20180363050A1 (en) 2015-12-07 2018-12-20 Raindance Technologies, Inc. Multiplexing in partitions using primer particles
WO2017100350A1 (en) 2015-12-07 2017-06-15 Raindance Technologies, Inc. Multiplexing in partitions using microparticles
WO2017117358A1 (en) 2015-12-30 2017-07-06 Bio-Rad Laboratories, Inc. Digital protein quantification
US10526605B2 (en) 2016-06-03 2020-01-07 Purdue Research Foundation siRNA compositions that specifically downregulate expression of a variant of the PNPLA3 gene and methods of use thereof for treating a chronic liver disease or alcoholic liver disease (ALD)
US20180057868A1 (en) 2016-08-30 2018-03-01 Integrated Dna Technologies, Inc. Cleavable hairpin primers

Non-Patent Citations (816)

* Cited by examiner, † Cited by third party
Title
Abate, 2011, Synthesis of monidisperse microparticles from non-Newtonian polymer solutions with microfluidic devices, Adv Mat 23(15):1757-1760.
Adang, 2001, The contribution of combinatorial chemistry to lead generation: an interim analysis, Curr Med Chem 8:985-998.
Advisory Action dated Sep. 9, 2014 for U.S. Appl. No. 13/679,190.
Affholter 1999, Engineering a Revolution, Chemistry in Britain 48-51.
Agrawal, 1990, Site-specific functionalization of oligodeoxynucleotides for non-radioactive labelling, Tetrahedron Let 31:1543-1546.
Aharoni, 2005, High-Throughput screens and selections of enzyme-encoding genes, Curr Opin Chem Biol, 9(2):210-6.
Ahn, 2006, Dielectrophoretic manipulation of drops for high-speed microluidic sorting devices, Applied Phys Lett 88:024104.
Akasheh, 2004, Development of piezoelectric micromachined ultrasonic transducers, Sensors and Actuators A Physical, 111:275-287.
Allen, 2000, High throughput fluorescence polarization: a homogeneous alternative to radioligand binding for cell surface receptors J Biomol Screen. 5(2):63-69.
Ammar, 2003, UV/Vis absorption and fluorescence spectroscopic study of novel symmetrical biscoumarin dyes, Dyes and Pigments 57:259-265.
Amplicon Sequencing, Application Note No. 5., Feb. 2007.
Amstutz, 2001, In vitro display technologies: novel developments and applications. Curr Opin Biotech 12:400-405.
Anarbaev, 1998, Klenow fragment and DNA polymerase alpha-primase fromserva calf thymus in water-in-oil microemulsions, Biochim Biophy Acta 1384:315-324.
Anderson, 1983, Preparation of a cell-free protein-synthesizing system from wheat germ, Methods Enz 101:635-644.
Anderson, 1993, Restriction endonucleases and modification methylases, Curr Op Struct Biol 3:24-30.
Ando, 1999, PLGA microspheres containing plasmid DNA: preservation of supercoiled DNA via cryopreparation and carbohydrate stabilization, J Pharm Sci 88(1):126-130.
Angell, 1983, Silicon micromechanical devices, Scientific Am 248:44-55.
Anhuf, 2003, Determination of SMN1 and SMN2 copy number using TaqMan technology, Hum Mutat 22(1):74-78.
Anna, 2003, Formation of dispersions using flow focusing in microchannels, Appl Phys Lett82(3):364-366.
Armstrong, 1996, Multiple-Component condensation strategies for combinatorial library synthesis, Acc Chem Res 29 (3):123-131.
Ashkin, 1987, Optical trapping and manipulation of single cells using infrared laser beams, Nature 330:769-771.
Ashkin, 1987, Optical trapping and manipulation of viruses and bacteria, Science 235(4795):1517-20.
Ashutosh Shastry et al., Directing Droplets Using Microstructured Surfaces.
Auroux, 2002, Micro Total Analysis Systems 2: Analytical standard operations and applications, Anal Chem 74 (12):2637-2652.
Baccarani, 1977, Escherichia coli dihydrofolate reductase: isolation and characterization of two isozymes, Biochemistry 16(16):3566-72.
Bagwe, 2001, Improved drug delivery using microemulsions: rationale, recent progress, and new horizons, Crit Rev Ther Drug Carr Sys 18(1):77-140.
Baker, 2010, Clever PCR: more genotyping, smaller volumes, Nat Meth 7:351-356.
Ballantyne, 1973, Selective area metallization by electron-beam controlled direct metallic deposition, J Vac Sci Tech 10:1094.
Barany, 1991, Genetic disease detection and DNA amplification using cloned thermostable ligase, PNAS 88(1):189-93.
Barany, 1991, The ligase chain reaction in a PCR World, PCR Meth App 1(1):5-16.
Baret, 2009, Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity, Lab Chip 9:1850-1858.
Baret, 2009, Kinetic aspects of emulsion stabilization by surfactants: a microfluidic analysis, Langmuir 25:6088-6093.
Baroud, 2004, Multiphase flows in microfluidics, Physique 5:547-555.
Bauer, 1999, Advances in cell separation: recent developments in counterflow centrifugal elutriation and continuous flow cell separation, J Chromot 722:55-69.
Beebe, 2000, Functional hydrogel structures for autonomous flow control inside microfluidic channels, Nature 404:588-590.
Beer, 2008, On-chip single-copy real-time reverse transcription PCR in isolated picoliter droplets, Anal Chem 80 (6):1854-1858.
Bein, 1999, Efficient assays for combinatorial methods for the eiscovery of catalysts, Agnew Chem Int Ed 38:3:323-26.
Benhar, I, et al., Highly efficient selection of phage antibodies mediated by display of antigen as Lpp-OmpA′ fusions on live bacteria, Journal of Molecular Biology, 301 893-904 (2000).
Benichou, 2002, Double emulsions stabilized by new molecular recognition hybrids of natural polymers, Polym Adv Tech 13:1019-1031.
Benner, 1994, Expanding the genetic lexicon, Trends Biotech 12:158-63.
Benning, 2000, The binding of substrate analogs to phosphotriesterase. J Biol Chem 275:30556-30560.
Berman, 1987, An agarose gel electrophoresis assay for the detection of DNA-binding activities in yeast cell extracts, Meth Enz 155:528-37.
Bernath, 2004, In Vitro Compartmentalization by double emulsions: sorting and gene enrichment by FACS Anal Biochem 325:151-157.
Bernath, 2005, Directed evolution of protein inhibitors of DNA-nucleases by in vitro compartmentalization (IVC) and nano-droplet delivery, J Mol Biol 345(5):1015-26.
Betlach, 1976, A restriction endonuclease analysis of the bacterial plasmid controlling the EcoRI restriction and modification of DNA, Fed Proc 35:2037-2043.
Bhagat et al., "Re-usable quick-release interconnect for characterization of microfluidic systems," J. Micromech. Microeng., vol. 17, pp. 42-49. (Year: 2007). *
Bhagat et al., 2007, Reusable quick-release interconnect for characterization of microfluidic systems, J Micromech Microeng 17:42-49.
Bibette, 1999, Emulsions: basic principles, Rep Prog Phys 62:969-1033.
Bico, 2002, Rise of liquids and bubbles in angular capillary tubes, J Colloid & Interface Sc 247:162-166.
Bico, 2002, Self-Propelling Slugs, J Fluid Mech 467:101-127.
Binder et al., Mismatch and G-stack modulated probe signals on SNP microarrays, PLoS One. Nov. 17, 2009;4(11): e7862.
Binladen, 2007, The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing, PLoSOne 2(2):e197.
Blanchet, 1993, Laser Ablation and the Production of Polymer Films, Science, 262(5134):719-721.
Boder, 1997, Yeast surface display for screening combinatorial polypeptide libraries, Nat Biotech 15(6):553-7.
Bosque, 2009, Induction of HIV-1 latency and reactivation in primary memory CD4+ T cells, Blood, 113(1):58-65.
Bougueleret, 1984, Characterization of the gene coding for the EcoRV restriction and modification system of E coli, Nucleic Acids Res 12(8):3659-76.
Branebjerg, 1996, Fast mixing by lamination, MEMS Proc 9th Ann 9:441-446.
Braslavsky, 2003, Sequence information can be obtained from single DNA molecules, PNAS 100(7):3960-3964.
Breslauer, 2006, Microfluidics based systems biology, Mol Bio Syst 2:97-112.
Bringer, 2004, Microfluidic systems for chemical kinetics that rely on chaotic mixing in droplets, Phil Trans A Math Phys Eng Sci 362:1-18.
Brown, 1979, Chemical synthesis and cloning of a tyrosine tRNA gene, Methods Enzymol 68:109-151.
Bru, 1991, Product inhibition of alpha-chymotrypsin in reverse micelles. Eur J Biochem 199(1):95-103.
Bru, 1993, Catalytic activity of elastase in reverse micelles, Biochem Mol Bio Int, 31(4):685-92.
Brummelkamp, 2002, A system for stable expression of short interfering RNAs in mammalian cells, Science 296 (5567):550-3.
Buican, 1987, Automated single-cell manipulation and sorting by light trapping, Appl Optics 26(24):5311-5316.
Burbaum, 1998, Miniaturization technologies in HTS, Drug Disc Today 3:313-322.
Burns, 1996, Microfabricated structures for integrated DNA analysis, PNAS 93:5556-5561.
Burns, 1998, An integrated nanoliter DNA analysis device, Science 282:484-487.
Burns, 2002, The intensification of rapid reactions in multiphase systems using slug flow in capillaries, Lab on a Chip 1:10-15.
Byrnes, 1982, Sensitive fluorogenic substrates for the detection of trypsin-like proteases and pancreatic elastase, Anal Biochem 126:447.
Cahill, 1991, Polymerase chain reaction and Q beta replicase amplification, Clin Chem 37(9):1482-5.
Caldwell, 1991, Limits of diffusion in the hydrolysis of substrates by the phosphodiesterase from Pseudomonas diminuta, Biochem 30:7438-7444.
Calvert, 2001, Inkjet printing for materials and devices, Chem Mater 13:3299-3305.
Caruccio, 2009, Nextura technology for NGS DNA library preparation: simulaneous fragmentation and tagging by in vitro transposition, Epibio Newsletter.
Caruthers, 1985, Gene synthesis machines: DNA chemistry and its uses, Science 230:281-285.
Cavalli, 2010, Nanosponge formulations as oxygen delivery systems, Int J Pharmaceutics 402:254-257.
Chakrabarti, 1994, Production of RNA by a polymerase protein encapsulated within phospholipid vesicles, J Mol Evol 39(6):555-9.
Chamberlain, 1973, Characterization of T7-specific ribonucleic acid polymerase. 1. General properties of the enzymatic reaction and the template specificity of the enzyme, J Biol Chem 248:2235-44.
Chan, 2003, Size-Controlled Growth of CdSe Nanocrystals in Microfluidic Reactors, Nano Lett 3(2):199-201.
Chan, 2008, New trends in immunoassays, Adv Biochem Engin/Biotech 109:123-154.
Chang, 1987, Recycling of NAD(P) by multienzyme systems immobilized by microencapsulation in artifical cells, Methods Enzymol, 136(67):67-82.
Chang, 2008, Controlled double emulsification utilizing 3D PDMS microchannels, Journal of Micromechanics and Microengineering 18:1-8.
Chao, 2004, Control of Concentration and vol. Gradients in Microfluidic Droplet Arrays for Protein Crystallization Screening, 26th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, San Francisco, California Sep. 1-5.
Chao, 2004, Droplet Arrays in Microfluidic Channels for Combinatorial Screening Assays, Hilton Head: A Solid State Sensor, Actuator and Microsystems Workshop, Hilton Head Island, South Carolina, Jun. 6-10.
Chapman, 1994, In vitro selection of catalytic RNAs, Curr. op. Struct. Biol., 4:618-22.
Chayen, 1999, Crystallization with oils: a new dimension in macromolecular crystal growth Journal of Crystal Growth, 196:434-441.
Chen, 2001, Capturing a Photoexcited Molecular Structure Through Time-Domain X-ray Absorption Fine Structure, Science 292(5515):262-264.
Chen, 2003, Microfluidic Switch for Embryo and Cell Sorting The 12th International Conference on Solid State Sensors, Actuators, and Microsystems, Boston, MA, Transducers, 1: 659-662.
Cheng, 2003, Electro flow focusing inmicrofluidic devices, Microfluidics Poster, presented at DBAS, Frontiers in Nanoscience, 1 page.
Cheng, 2006, Nanotechnologies for biomolecular detection and medical diagnostics, Current Opinion in Chemical Biology, 10:11-19.
Chen-Goodspeed, 2001, Enhancement, relaxation, and reversal of the stereoselectivity for phosphotriesterase by rational evolution of active site residues, Biochemistry, 40: 1332-1339.
Chen-Goodspeed, 2001, Structural Determinants of the substrate and stereochemical specificity of phosphotriesterase, Biochemistry, 40(5):1325-31.
Chetverin, 1995, Replicable RNA vectors: prospects for cell-free gene amplification, expression, and cloning, Prog Nucleic Acid Res Mol Biol, 51:225-70.
Chiang, 1993, Expression and purification of general transcription factors by FLAG epitope-tagging and peptide elution, Pept Res, 6:62-64.
Chiba, 1997, Controlled protein delivery from biodegradable tyrosino-containing poly(anhydride-co-imide) microspheres, Biomaterials, 18(13):893-901.
Chiou, 2001, A closed-cycle capillary polymerase chain reaction machine, Analytical Chemistry, American Chamical Society, 73:2018-21.
Chiu, 1999, Chemical transformations in individual ultrasmall biomimetic containers, Science, 283:1892-1895.
Chiu, 2008, Noninvasive prenatal diagnosis of chromosomal aneuploidy by massively paralel genomic seuqencing of DNA in maternal plasma, PNAS 105(51):20458-20463.
Chou, 1998, A microfabricated device for sizing and sorting DNA molecules 96:11-13.
Clackson, 1994, In vitro selection from protein and peptide libraries, Trends Biotechnol, 12:173-84.
Clausell-Tormos, 2008, Droplet-based microfluidic platforms for the encapsulation and screening of Mammalian cells and multicellular organisms, Chem Biol 15(5):427-437.
Cohen, 1991, Controlled delivery systems for proteins based on poly(lactic/glycolic acid) microspheres, Pharm Res, 8 (6):713-720.
Collins, 2003, Optimization of Shear Driven Droplet Generation in a Microluidic Device, ASME International Mechanical Engineering Congress and R&D Expo, Washington, 4 pages.
Collins, 2004, Microfluidic flow transducer based on the measurements of electrical admittance, Lab on a Chip, 4:7-10.
Compton, 1991, Nucleic acid sequence-based amplification, Nature, 350(6313):91-2.
Cook, 2007, Use and misuse of receiver operating characteristic curve in risk prediction, Circulation 115(7):928-35.
Cooper, 2000, The Central Role of Enzymes as Biological Catalysts, The Cell: A Molecular Approach, 2nd Edition, pp. 1-6.
Cormack, 1996, FACS-optimized mutants of the green fluorescent protein (GFP), Gene 173(1):33-38.
Cortesi, 2002, Production of lipospheres as carriers for bioactive compounds, Biomateials, 23(11): 2283-2294.
Courrier, 2004, Reverse water-in-fluorocarbon emulsions and microemulsions obtained with a fluorinated surfactant, Colloids and Surfaces A: Physicochem. Eng. Aspects 244:141-148.
Craig, 1995, Fluorescence-based enzymatic assay by capillary electrophoresis laser-induced fluoresence detection for the determinination of a few alpha-galactosidase molecules, Anal. Biochem. 226:147.
Creagh, 1993, Structural and catalytic properties of enzymes in reverse micelles, Enzyme Microb Technol 15 (5):383-92.
Crosland-Taylor, 1953, A Device for Counting Small Particles suspended in a Fluid through a Tube, Nature 171:37-38.
Crowley, 1973, Electrical breakdown of bimolecular lipid membranes as an electromechanical instability, Biophys J. 13 (7):711-724.
Cull, 1992, Screening for receptor ligands using large libraries of peptides linked to the C terminus of the lac repressor, PNAS 89:1865-9.
Curcio, 2003, Continuous Segmented-Flow Polymerase Chain Reaction for High-Throughput Miniaturized DNA Amplification, Anal Chem 75(1):1-7.
Curran, 1998, Strategy-level separations in organic synthesis: from planning to practice. Angew Chem Int Ed, 37:1174-11-96.
Czarnik, 1997, Encoding methods for combinatorial chemistry, Curr Opin Chem Biol 1:60-66.
Dankwardt, 1995, Combinatorial synthesis of small-molecule libraries using 3-amino-5-hydroxybenzoic acid, 1:113-120.
David, 1974, Protein iodination with solid-state lactoperoxidase, Biochemistry 13:1014-1021.
Davies, 1997, Activated Alzheimer Disease Platelets Retain More Beta Amyloid Precursor Protein, Neurobiology of Aging, 18(2):147-153.
Davis, 1987, Multiple emulsions as targetable delivery systems, Meth Enzymol 149:51-64.
Davis, 2006, Deterministic hydrodynamics: Taking blood apart, PNAS 103:14779-14784.
De Gans, 2004, Inkjet printing of polymers: state of the art and future developments, Advanced materials, 16: 203-213.
De Wildt, 2002, Isolation of receptor-ligand pairs by capture of long-lived multivalent interaction complexes, Proceedings of the National Academy of Sciences of the United States, 99, 8530-8535.
De Wildt, Ruud, et al., Isolation of receptor-ligand pairs by capture of long-lived multivalent interaction complexes, Proceedings of the National Academy of Sciences of the United States, 99, 8530-8535 (2002).
Definition of Nozzle [retrieved on-line, retrieval date, Jun. 26, 202, retrieved from http://www.merriam-webster.com/dictionary/nozzle (Year: 2020). *
DelRaso, 1993, In vitro methodologies for enhanced toxicity testing, Toxicol. Lett. 68:91-99.
Deng et al., Design and analysis of mismatch probes for long oligonucleotide microarrays, BMC Genomics. 2008; 9:491.
Dickinson, 1992, Interfacial interactions and the stability of oil-in-water emulsions, Pure Appl Chem 64(11):1721-1724.
Dickinson, 1994, Emulsions and droplet size control, Wedlock, D.J., Ed., in Controlled Particle Droplet and Bubble Formulation, ButterWorth-Heine-mann, 191-257.
DiMatteo, 2008, Genetic conversion of an SMN2 gene to SMN1: A novel approach to the treatment of spinal muscular atrophy, Exp Cell Res. 314(4):878-886.
Ding, 2001, Scheduling of microfluidic operations for reconfigurable two-dimensional electrowetting arrays, IEEE Trans CADICS 20(12):1463-1468.
Ding, Proc. Natl. Acad. Sci. USA, 2003, 100(33):7449-7453.
Dinsmore, 2002, Colioidosomes: Selectively Permeable Capsules Composed of Colloidal Particles, Science 298 (5595):1006-1009.
Dittrich, 2005, A new embedded process for compartmentalized cell-free protein expression and on-line detection in microfluidic devices, Chembiochem 6(5):811-814.
Doi, 1999, Stable: protein-DNA fusion system for screening of combinatorial protein libraries in vitro, FEBS Lett., 457: 227-230.
Doi, 2004, In vitro selection of restriction endonucleases by in vitro compartmentilization, Nucleic Acids Res, 32(12): e95.
Doman, 2002, Molecular docking and high-throughput screening for novel inhibitors of protein tyrosine phosphatase-1B, J Med Chem, 45: 2213-2221.
Domling, 2000, Multicomponent Reactions with Isocyanides, Angew Chem Int Ed 39(18):3168-3210.
Domling, 2002, Recent advances in isocyanide-based multicomponent chemistry, Curr Opin Chem Biol, 6(3):306-13.
Dorfman et al., "Contamination-Free Continuous Flow Microfluidic Polymerase Chain Reaction for Quantitative and Clinical Applications," Analytical Chemistry, vol. 77, pp. 3700-3704. (Year: 2005). *
Dorfman et al., 2005, Contamination-Free Continuous Flow Microfluidic Polymerase Chain Reaction for Quantitative and Clinical Applications, Anal Chem 77(11):3700-04.
Dorfman, 2005, Contamination-free continuous flow microfluidic polymerase chain reaction for quantitative and clinical applications, Anal Chem 77:3700-3704.
Dove, 2002, Research News Briefs, Nature Biotechnology 20:1213, 1 page.
Dower, 1988, High efficiency transformation of E. coli by high voltage electroporation, Nucleic Acids Res 16:6127-6145.
Dressman, 2003, Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations, PNAS 100:8817-22.
Dreyfus, 2003, Ordered and disordered patterns in two phase flows in microchannels, Phys Rev Lett 90 (14):144505-1-144505-4.
Drmanac, 1992, Sequencing by hybridization: towards an automated sequencing of one million M13 clones arrayed on membranes, Elctrophoresis 13:566-573.
Du, Wenbin, et al., SlipChip, Lab Chip, 2009, 9, 2286-2292.
Dubertret, 2002, In vivo imaging of quantum dots encapsulated in phospholipid micelles, Science, 298: 1759-1762.
Duffy, 1998, Rapid Protyping of Microfluidic Systems and Polydimethylsiloxane, Anal Chem 70:474-480.
Duggleby, 1995, Analysis of Enzyme Progress Curves by Nonlinear Regression, Pt D. Academic Press 249:61-90.
Dumas, 1989, Purification and properties of the phosphotriesterase from Psuedomonas diminuta, J Biol Chem 264: 19659-19665.
Eckert, 1991, DNA polymerase fidelity and the polymerase chain reaction, Genome Res 1:17-24.
Ecole Polytech Federale de Lausanne, 2014, Tracing water channels in cell surface receptors, PhysOrg News (2 pages).
Edel, 2002, Microfluidic Routes to the Controlled Production of Nanopaticles, Chemical Communications, 1136-1137.
Edris, 2001, Encapsulation of orange oil in a spray dried double emulsion, Nahrung/Food, 45(2):133-137.
Effenhauser, 1993, Glass chips for high-speed capillary electrophoresis separations with submicrometer plate heights, Anal Chem 65:2637-2642.
Eggers, 1999, Coalescence of Liquid Drops, J Fluid Mech 401:293-310.
Ehrig, 1995, Green-fluorescent protein mutants with altered fluorescence excitation spectra, Febs Lett, 367(2):163-66.
Eigen, 1980, Hypercycles and compartments: compartments assists—but does not replace—hypercyclic organization of early genetic information, J Theor Biol, 85:407-11.
Eijk-Van Os, 2011, Multiplex ligation-dependent probe amplification (MLPA(R)) for the detection of copy number variation in genomic sequences, Meth Mol Biol 688:97-126.
Elghanian, 1997, Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles, Science, 277(5329):1078-1080.
Ellington, 1990, In vitro selection of RNA molecules that bind specific ligands, Nature, 346:818-822.
Ellman, 1991, Biosynthetic method for introducing unnatural amino acids site-specifically into proteins, Methods Enzymol, 202:301-36.
Endo, 1996, Autocatalytic decomposition of cobalt complexes as an indicator system for the determination of trace amounts of cobalt and effectors, Analyst 121:391-394.
Endo, 1998, Kinetic determination of trace cobalt by visual autocatalytic indication, Talanta 47:349-353.
Engl, 2005, Droplet Traffic at a Simple Junction at Low Capillary Numbers Physical Review Letters, vol. 95, 208304, 1 page.
Engl, W. et al, Droplet Traffic at a Simple Junction at Low Capillary Numbers Physical Review Letters, 2005, vol. 95,208304.
Eow, 2002, Electrocoalesce-separators for the separation of aqueous drops from a flowing dielectric viscous liquid, Separation and Purification Tech 29:63-77.
Eow, 2002, Electrostatic and hydrodynamic separation of aqueous drops in a flowing viscous oil, Chemical Eng Proc 41:649-657.
Eow, 2002, Electrostatic enhancement of coalescence of water droplets in oil: a review of the technology, Chemical Engineeing Journal 85:357-368.
Eow, 2003, Motion, deformation and break-up of aqueous drops in oils under high electric field strengths, Chemical Eng Proc 42:259-272.
Eow, 2003, The behavior of a liquid-liquid interface and drop-interface coalescence under the influence of an electric field, Colloids and Surfaces A: Physiochern. Eng. Aspects 215:101-123.
Eriksson, 2013, Comparative analysis of measures of viral reservoirs in HIV-1 eradication studies, PLoS Pathogens 9 (2):e1003174, 17 pages.
European Office Action dated Apr. 29, 2014 for EP 08165420.4.
European Search Report for EP 13165665.4 mailed Nov. 22, 2013, 4 pages.
European Search Report for EP 13165667.0 mailed Nov. 22, 2013, 4 pages.
European Search Report for EP Application No. 13165665 with the date of the completion of the search Nov. 15, 2013 (4 pages).
European Search Report for EP Application No. 13165667 with the date of the completion of the search Nov. 15, 2013 (4 pages).
Extended European Search Report for EP 10196179 mailed May 2, 2014 (10 pages).
Extended European Search Report for EP 10196339 mailed May 2, 2014 (9 pages).
Faca, 2008, A mouse to human search for plasma proteome changes associated with pancreatic tumor development, PLoS Med 5(6):el23:0953-0967.
Fahy, 1991, Self-sustained sequence replication (3SR): an isothermal transcription-based amplification system alternative to PCR, PCR Methods Appl 1:25-33.
Fan et al., Detection of Aneuploidy with Digital PCR, available at https://arxiv.org/ftp/arxiv/papers /0705/0705.1 030.pdf, Dec. 31, 2007.
Fan, 1994, Micromachining of capillary electrophoresis injectors and separators on glass chips and evaluation of flow at capillary intersections, Anal Chem 66:177-184.
Fastrez, 1997, In vivo versus in vitro screening or selection for catalytic activity in enzymes and abzymes, Mol Biotechnol 7(1):37-55.
Fettinger, 1993, Stacked modules for micro flow systems in chemical analysis: concept and studies using an enlarged model, Sens Actuat B. 17:19-25.
Fiedler, 1998, Dielectrophoretic sorting of particles and cells in a microsystem, Anal Chem 70(9):1909-1915.
Field, 1988, Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cervisiae by use of an epitope addition method. Mol Cell Biol, 8: 2159-2165.
Fields, 1989, A novel genetic system to detect protein-protein interactions, Nature 340(6230):245-6.
Filella, 1994, TAG-72, CA 19.9 and CEA as tumor markers in gastric cancer, Acta Oncol. 33(7):747-751.
Finch, 1993, Encapsulation and controlled release, Spec Publ R Soc Chem, 138:35, 12 pages.
Fingas, 1997, Studies of Water-In-Oil Emulsions: Stability Studies, Environment Canada, Proceedings of the Twentieth Arctic Marine Oilspill Program Technical Seminer, 1-20.
Fire, 1995, Rolling replication of short DNA circles, PNAS 92(10):4641-5.
Firestine, 2000, Using an AraC-based three hybrid system to detect biocatalysts in vivo, Nat Biotechnol 18: 544-547.
Fisher, 2004, Cell Encapsulation on a Microfluidic Platform, The Eighth International Conference on Miniaturised Systems for Chemistry and Life Scieces, MicroTAS, Malmo, Sweden.
Fletcher, 2002, Micro reactors: principles and applications in organic synthesis, Tetrahedron 58:4735-4757.
Fluri, 1996, Integrated capillary electrophoresis devices with an efficient postcolumn reactor in planar quartz and glass chips, Anal Chem 68:4285-4290.
Fornusek, 1986, Polymeric microspheres as diagnostic tools for cell surface marker tracing, Crit Rev Ther Drug Carrier Syst, 2:137-74.
Fowler, 2002, Enhancement of Mixing By Droplet-Based Microfluidics, Int Conf MEMS 97-100.
Frenz, 2008, Reliable microfluidic on-chip incubation of droplets in delay-lines, Lab on a Chip 9:1344-1348.
Fu, 1999, A microfabricated fluorescence-activated cell sorter, Nature Biotechnology, 17(11):1109-1111.
Fu, 2002, An Integrated Microfabricated Cell Sorter, Anal. Chem., 74: 2451-2457.
Fulton, 1997, Advanced multiplexed analysis with the FlowMetrix system, Clin Chem 43:1749-1756.
Fulwyler, 1965, Electronic Separation of Biological Cells by Volume, Science 150(3698):910-911.
Fungi (Wikipedia.com accessed Jun. 3, 2013).
Galan, 2010, A 454 multiplex sequencing method for rapid and reliable genotyping of highly polymorphic genes in large-scale studies., BMC Genomics 11(296):1-15.
Gallarate, 1999, On the stability of ascorbic acid in emulsified systems for topical and cosmetic use, Int J Pharm 188 (2):233-241.
Ganan-Calvo, 1998, Generation of Steady Liquid Microthreads and Micron-Sized Monodisperse Sprays and Gas Streams, Phys Rev Lett 80(2):285-288.
Ganan-Calvo, 2001, Perfectly Monodisperse Microbubbling by Capillary Flow Focusing, Phys Rev Lett 87(27): 274501-1-4.
Garcia-Ruiz, 1994, Investigation on protein crystal growth by the gel acupuncture method, Acta, Cryst., D50, 99. pp. 484-490.
Garcia-Ruiz, 2001, A super-saturation wave of protein crystallization, J. Crystal Growth, 232:149-155.
Garstecki, 2004, Formation of monodisperse bubbles in a microfiuidic flow-focusing device, Appl Phys Lett 85 (13):2649-2651.
Gasperlin, 1994, The structure elucidation of semisolid w/o emulsion systems containing silicone surfactant, Intl J Pharm, 107:51-6.
Gasperlin, 2000, Viscosity prediction of lipophilic semisolid emulsion systems by neural network modeling, Intl J Pharm, 196:37-50.
Gelderblom, 2008, Viral complemntation allows HIV-1 replication without integration, Retrovirology 5:60.
Georgiou, 1997, Display of heterologous proteins on the surface of microorganisms: from the screenign of combinatiorial libraires to live recombinant vaccines. Nat Biotechnol 15(1), 29-34.
Georgiou, 2000, Analysis of large libraries of protein mutants using flow cytometry, Adv Protein Chem, 55: 293-315.
Gerdts, 2004, A Synthetic Reaction NetWork: Chemical Amplification Using Nonequilibrium Autocatalytic Reactions Coupled in Time, J. Am. Chem. Soc 126:6327-6331.
Ghadessy, 2001, Directed Evolution of Polymerase Function by Compartmentalized Self-Replication, PNSAS 98(8): 4552-4557.
Gibbs, 1989, Detection of single DNA base differences by competitive oligonucleotide priming, Nucleic Acids Res. 17 (7): 2437-48.
Gilliland, 1990, Analysis of cytokine mRNA and DNA: Detection and quantitation by competitive polymerase chain reaction, PNAS, 87(7):2725-9.
Giusti, 1993, Synthesis and characterization of 5′ fluorescent dye labeled oligonucleotides, Genome Res 2:223-227.
Glass, 1995, Development of primer sets designed for use with the PCR to amlify conserved genes from filamentous ascomycetes, Applied and Environmental Microbiology, vol. 6, pp. 1323-1330.
Gold, 1995, Diversity of Oligonucleotide Functions Annu Rev Biochem, 64: 763-97.
Gong, 2015, Simple method to prepare oligonucleotide conjugated antibodies and its applicaiotn in multiplex protein detection in single cells, Bioconjugate Chm 27(1):271-225.
Goodall, 1998, Operation of Mixed-Culture Immobilized Cell Reactors for the Metabolism of Meta- and Para-Nitrobenzoate by Comamonas Sp. JS46 and Comamonas Sp. JS47, Biotechnology and Bioengineering, 59 (1): 21-27.
Gordon, 1999, Solid phase synthesis—designer linkers for combinatorial chemistry: a review, J. Chem. Technol. Biotechnol., 74(9):835-851.
Grasland-Mongrain, 2003, Droplet coalescence in microfluidic devices, 30 pages, From internet: http://www.eleves.ens.fr/home/grasland/rapports/stage4.pdf.
Gray, 1987, High speed crhomosome sorting, Science 238(4825):323-329.
Green, 1992, Selection of a Ribozyme That Functions as a Superior Template in a Self Copying Reaction, Science, 258: 1910-5.
Gregoriadis, 1976, Enzyme entrapment in liposomes, Methods Enzymol 44:218-227.
Griffiths, 2000, Man-made enzymes-from design to in vitro compartmentalisation, Curr Opin Biotechnol 11:338-353.
Griffiths, 2003, Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization, EMBO J, 22:24-25.
Griffiths, 2006, Miniaturising the laboratory in emulsion droplets, Trend Biotech 24(9):395-402.
Grinwood, 2004, The DNA sequence and biology of human chromosome 19, Nature 428:529-535.
Grothues, 1993, PCR amplification of megabase DNA with tagged random primers (T-PCR), Nucl. Acids Res vol. 21 (5):1321-1322.
Grund, 2010, Analysis of biomarker data:logs, odds, ratios and ROC curves, Curr Opin HIV AIDS 5(6):473-479.
Gruner, 2015, Stabilisers for water-in-fluorinated-oil dispersions, Curr Op Coll Int Sci 20:183-191.
Guatelli, 1990, Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication, PNAS, 87(5):1874-8.
Guixe, 1998, Ligand-Induced Conformational Transitions in Escherichia coli Phosphofructokinase 2: Evidence for an Allosteric Site for MgATP2n, Biochem., 37: 13269-12375.
Guo, 2010, Simultaneous detection of trisomies 13, 18, and 21 with multiplex ligation dependent probe amplification-based real-time PCR, Clin Chem 56(9):1451-1459.
Gupta, 1991, A general method for the synthesis of 3′-sulfhydryl and phosphate group containing oligonucleotides, Nucl Acids Res 19 (11): 3019-3026.
Haber, 1993, Activity and spectroscopic properties of bovine liver catalase in sodium bis(2-ethylhexyl) sulfosuccinate/isooctane reverse micelles, Eur J Biochem 217(2): 567-73.
Habig, 1981, Assays for differentiation of glutathione S-transferases, Methods in Enzymology, 77: 398-405.
Hadd, 1997, Microchip Device for Performing Enzyme Assays, Anal. Chem 69(17): 3407-3412.
Haeberle, 2007, Microfluidic platforms for lab-on-a-chip applications, Lab on a Chip 7:1081-1220.
Hagar, 1992, The effect of endotoxemia on concanavalin A induced alterations in cytoplasmic free calcium in rat spleen cells as determined with Fluo-3, Cell Calcium 13:123-130.
Hai, 2004, Investigation on the release of fluorescent markers from the w/o/w emulsions by fluorescence-activated cell sorter, J Control Release, 96(3): 393-402.
Haies, 1981, Morphometric study of rat lung cells. I. Numerical and dimensional characteristics of parenchymal cell population, Am. Rev. Respir. Dis. 123:533-54.
Hall, 2003, The EBG system of E. coli: origin and evolution of a novel beta-galactosidase for the metabolism of lactose, Genetica 118(2-3):143-56.
Hamady, 2008, Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex. Nature Nethods vol. 5, No. 3, p. 235-237.
Han, 2001, Quantum-dot-tagged Microbeads for Multiplexed Optical Coding of Biomolecules, Nat Biotech 19(7): 631-635.
Handen, 2002, High-throughput screening—challenges for the future, Drug Discov World, 47-50.
Handique, 2001, On-Chip Thermopneumatic Pressure for Discrete Drop Pumping, Analytical Chemistry, 73:1831-1838.
Hanes, 1997, In vitro selection and evolution of functional proteins by using ribosome display, PNAS 94:4937-42.
Hanes, 1998, Degradation of porous poly(anhydide-co-imide) microspheres and implication for controlled macromolecule delivery, Biomaterials, 19(1-3): 163-172.
Hansen, 2002, A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion, PNAS 99(26):16531-16536.
Harada, 1993, Monoclonal antibody G6K12 specific for membrane-associated differentiation marker of human stratified squamous epithelia and squamous cell carcinoma, J. Oral Pathol. Med 22(4):145-152.
Harder, 1994, Characterization and kinetic analysis of the intracellular domain of human protein tyrosine phosphatase beta (HPTP beta) using synthetic phosphopeptides, Biochem J 298 (Pt 2): 395-401.
Harries, 2006, A Numerical Model for Segmented Flow in a Microreactor, Int J of Heat and Mass Transfer, 46:3313-3322.
Harris, 2008, Single-molecule DNA sequencing of a viral genome, Science 320(5872):106-109.
Harrison, 1993, Micromachining a miniaturized capillary electrophoresis-based chemical analysis system on a chip, Science 261(5123):895-897.
Hasina, 2003, Plasminogen activator inhibitor-2: a molecular biomarker for head and neck cancer progression, Cancer Research 63:555-559.
Haynes, 2012, Digital PCR: A Technology Primer, Principles of Digital PCR and Measurement Issues: The certification of Cytomegalovirus Standard Reference Material (SRM 2366) as a model for future SRMs, National Institute of Standards and Tecnology, San Diego, CA, 4 pages.
Hayward, 2006, Dewetting Instability during the Formation of Polymersomes from BloceCopolymer-Stabilized Double Emulsions, Langmuir, 22(10): 4457-4461.
He, 2005, Selective encapsulation of single cells and subcellular organelles into picoliter- and femtoliter-volume droplets, Anal Chem 77(6):1539-1544.
Head, 2014, Library construction for next generation sequencing, Biotech Rap Disp 56(2):61.
Heim, 1996, Engineering Green Fluorescent Protein for Improved Brightness, Longer Wavelengths and Fluorescence Response Energy Transfer, Carr. Biol, 6(2): 178-182.
Hellman, 2009, Differential tissue-specific protein markers of vaginal carcinoma, Br J Cancer, 100(8): 1303-131.
Henrich, 2012, Low-level detection and quantitation of cellular HIV-1 DNA and 2-ILTR circles using droplet dPCR, J Virol Meth 186(1-2):68-72.
Hergenrother, 2000, Small-Molecule Microarrays: Covalent Attachment and Screening of Alcohol-Containing Small Molecules on Glass Slides, J. Am. Chem. Soc, 122: 7849-7850.
Hermankova, 2003, Analysis of human immunodeficiency virus type 1 gene expression in lately infected reseting CD4 T lymphocytes in vivo, J Virology 77(13):7383-7392.
Herzer, 2001, DNA Purification, in Molecular Biology Problem Solver: A Laboratory Guide, Edited by Alan S. Gerstein, Ch.1.
Heyries, Kevin A, et al., Megapixel digital PCR, Nat. Methods 8, 649-651 (2011).
Hildebrand, 1949, Liquid-Liquid Solubility of Perfluoromethylcyclohexane with Benzene, Carbon Tetrachloride, Chlorobenzene, Chloroform and Toluene, J. Am. Chem. Soc, 71: 22-25.
Hindson et al., 2011, High-Throughput Droplets Digital PCR System for Absolute Quantitation of DNA Copy Number, Anal Chem 83:8604-8610.
Hindson, Benjamin J., et al., High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number, Anal. Chem., 83, 8604-8610 (2011).
Hisu, 1999, et al., Comparison of process parameters for microencapsulation of plasmid DNA in poly(D, L-lactic-co-glycolic acid microspheres, J Drug Target, 7:313-23.
Hjelmfelt, 1993, Pattern-Recognition in Coupled Chemical Kinetic Systems, Science, 260(5106):335-337.
Ho, 1989, Site-directed mutageneiss by overlap extension using the polymerase chain reaction, Gene, 77(1):51-9.
Hochuli, 1987, New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues, J Chromatogr 411: 177-84.
Holmes, 1995, Reagents for Combinatorial Organic Synthesis: Development of a New O-Nitrobenzyl Photolabile Linder for Solid Phase Synthesis, J. OrgChem., 60: 2318-2319.
Holtze, C., et al., Biocompatible surfactants for water-in-fluorocarbon emulsions, Lab Chip, 2008, 8, 1632-1639.
Hong, 1999, Stereochemical constraints on the substrate specificity of phosphodiesterase, Biochemistry, 38: 1159-1165.
Hoogenboom, 1997, Designing and optimizing library selection strategies for generating high-affinity antibodies, Trends Biotechnol, 15:62-70.
Hopfinger, 1996, Explosive Breakup of a Liquid Jet by a Swirling Coaxial Jet, Physics of Fluids 8(7):1696-1700.
Hopman, 1998, Rapid synthesis of biotin-, digoxigenin-, trinitrophenyl-, and fluorochrome-labeled tyramides and their application for In situ hybridization using CARD amplification, J of Histochem and Cytochem, 46(6):771-77.
Horton, 1989, Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension, Gene 77(1):61-8 (.
Hosokawa, 1999, Handling of Picoliter Liquid Samples in a Poly(dimethylsiloxane)-Based Microfluidic Device, Analytical Chemistry, 71(20):4781-4785.
How many species of bacteria are there(wisegeek.com; accessed Sep. 23, 2011).
Hsieh, 2009, Rapid label-free DNA analysis in picoliter microfluidic droplets using FRET probes, Microfluidics and nanofluidics 6(3):391-401.
Hua, 2010, Multiplexed Real-Time Polymerase Chain Reaction on a Digital Microfluidic Platform, Analytical Chemistry 82(6):2310-2316.
Huang et al., Identification of 8 foodborne pathogens by multicolor combinational probe coding technology in a single real-time PCR, Clin Chem. Oct. 2007;53(10):1741-8.
Huang, 1991, Kinetic assay of fluorescein mono-beta-D-galactosidase hydrolysis by beta-galactosidase: a front-face measurement for strongly absorbing fluorogenic substrates, Biochemistry, 30:8530-4.
Huang, 1992, A sensitive competitive ELISA for 2,4-dinitrophenol using 3,6-fluorescein diphosphate as a fluorogenic substrate, J Immunol Meth, 149:261.
Huang, 2004, Continuous particle separation through deterministic lateral displacement, Science 304(5673):987-990.
Huang, 2007, Identification of 8 foodborne pathogens by multicolor combinational probe coding technology in a single real-time PCR, Clin Chem., 53(10):1741-8.
Hubert, 2003, Data Concordance from a Comparison between Filter Binding and Fluorescence Polarization Assay Formats for Identification of RUOCK-II Inhibitors, J biomol Screen 8(4):399-409.
Huebner, 2007, Quantitative detection of protein expression in single cells using droplet microfluidics, Chem Com 12:1218-1220.
Hug et al. Measurement of the number of molecules of a single mRNA species in a complex mRNA preparation. J Theor Biol.; 221(4):615-24 (2003).
Hug, 2003, Measurement of the number of molecules of a single mRNA species in a complex mRNA preparation. J Theor Biol.; 221(4):615-24.
Hung, 2004, Controlled Droplet Fusion in Microfluidic Devices, MicroTAS 2004, Sep. 26-30, Malmo, Sweden.
Hung, 2004, Optimization of Droplet Generation by controlling PDMS Surface Hydrophobicity, 2004 ASME International Mechanical Engineering Congress and RD&D Expo, Nov. 13-19, Anaheim, CA, 47-48.
Hutchison, 2005, Cell-free cloning using Phi29 polymerase, PNAS 102(48):17332-17336.
Ibrahim, 2003, High-speed cell sorting: fundamentals and recent advances, Curr Opin Biotchnol, 14(1):5-12.
Ikeda, 2000, Bioactivation of tegafur to 5-fluorouracil is catalyzed by cytochrome P-450 2A6 in human liver microsomes in vitro, Clin Cancer Res 6(11):4409-4415.
Ilumina, 2010, Genomic Sequencing, data Sheet, 6 pages.
Inai, 1993, Immunohistochemical detection of an enamel protein-related epitope in rat bone at an early stage of osteogenesis, Histochemistry 99(5):335-362.
International Preliminary Report on Patentability PCT/US2004/027912 dated Jan. 26, 2005, 7 pages.
International Search Report and Written Opinion for PCT/US2013/037751 dated Aug. 22, 2013.
International Search Report and Written Opinion mailed on Apr. 23, 2015, for International Patent Application No. PCT/US14/34205, filed Apr. 15, 2014, 18 pages.
International Search Report and Written Opinion mailed on Nov. 25, 2014, for International Patent Application No. PCT/US14/34037, filed Apr. 14, 2014, 13 pages.
International Search Report and Written Opinion, mailed Jun. 11, 2015, for PCT/US2014/032614, filed Apr. 2, 2014 (9 pages).
International Search Report for PCT/US2003/2052 dated Jun. 6, 2004.
International Search Report for PCT/US2006/001938 dated May 31, 2006, 5 pages.
Invitrogen, 2008, Specification sheet for Dynabeads® Oligo (dT)25, http://www.invitrogen.com, 2 pages.
Ismagilov, 2003, Integrated Microfluidic Systems, Angew. Chem. Int. Ed 42:4130-4132.
Jakobovits, 1993, Analysis of homozygous mutant chimeric mice:deletion of the immunoglobulin heavy-chain joining region blocks B-cell development and antibody production, PNAS USA 90:2551-255.
Jakobovits, 1993, Germ-line transmission and expression of a human-derived yeast artificial chromosome, Nature 362:255-258.
Janda, 1997, Chemical selection for catalysis in combinatorial antibody libraries, Science, 275:945-948.
Jang, 2003, Controllable delivery of non-viral DNA from porous scaffold, J Controlled Release 86(1):157-168.
Japanese Office Action for Application No. JP 2009-231040 mailed Jul. 1, 2014.
Japanese Office Action for JP 2006-509830 mailed Jun. 1, 2011 (4 pages).
Jermutus, 1998, et al., Recent advances in producing and selecting functional proteins by using cell-free translation, Curr Opin Biotechnol 9(5): 534-48.
Jo, 2003, Encapsulation of Bovine Serum Albumin in Temperature-Programmed Shell-in-Shell Structures, Macromol. Rapid Comm 24:957-962.
Joerger, 1995, Analyte detection with DNA-labeled antibodies and polymerase chain reaction, Clin. Chem. 41 (9):1371-7.
Johannsson, 1988, Amplification by Second Enzymes, In ELISA and Other Solid Phase Immunoassays, Kemeny et al (ed.), Chapter 4, pp. 85-106 John Wiley.
Johannsson, 1991, Heterogeneous Enzyme Immunoassays, In Principles and Practice of Immunoassay, pp. 295-325 Stockton Press.
Johnson, 1993, Human antibody engineering: Current Opinion in Structural Biology, 3:564-571.
Johnson, 2002, Protein tyrosine phosphatase 1B inhibitors for diabetes, Nature Review Drug Discovery 1, 696-709.
Jones, 1986, Replacing the complementarity-determining regions in a human antibody with those from a mouse, Nature, 321:522-525.
Jones, 1997, Quenched BODIPY dye-labeled casein substrates for the assay of protease activity by direct fluorescence measurement, Anal Biochem, 251:144-152.
Jones, 1999, Glowing jellyfish, luminescence and a molecule called coelenterazine, Trends Biotechnol. 17(12):477-81.
Joo, 1999, Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylaion, Nature 399:670.
Joos, 1997, Covalent attachment of hybridizable oligonucleotides to glass supports, Analytical Biochemistry 247:96-101.
Joyce,1994, In vitro Evolution of Nucleic Acids, Curr. Opp. Structural Biol, 4: 331-336.
Kadir, 1990, Haem binding to horse spleen ferritin, Febs Lett, 276: 81-4.
Kallen, 1966, The mechanism of the condensation of formaldehyde with tetrahydrofolic acid, J. Biol. Chem., 241:5851-63.
Kambara, 1988, Optimization of Parameters in a DNA Sequenator Using Fluorescence Detection, Nature Biotechnology 6:816-821.
Kamensky, 1965, Spectrophotometer: new instrument for ultrarapid cell analysis, Science 150(3696):630-631.
Kanouni, 2002, Preparation of a stable double emulsion (W1/0/W2): role of the interfacial films on the stability of the system, Adv. Collid. Interf. Sci., 99(3): 229-254.
Karapatis, 1998, Direct rapid tooling:a review of current research, Rapid Prototyping Journal, 4(2):77-89.
Katanaev, 1995, Viral Q beta RNA as a high expression vector for mRNA translation in a cell-free system, Febs Lett, 359:89-92.
Katsura, 2001, Indirect micromanipulation of single molecules in water-in-oil emulsion, Electrophoresis, 22:289-93.
Kawakatsu, 1997, Regular-sized cell creation in microchannel emulsification by visual microprocessing method, Journal of the American Oil ChemistS Society, 74:317-21.
Keana, 1990, New reagents for photoaffinity labeling: synthesis and photolysis of functionalized perfluorophenyl azides, J. Org. Chem.55(11):3640-3647.
Keefe, 2001, Functional proteins from a random-sequence library, Nature, 410: 715-718.
Keij, 1994, High-speed photodamage cell sorting: An evaluation of the ZAPPER prototype, Methods in cell biology, 42: 371-358.
Kelly, 2005, Detection of Vascular Adhesion Molecule-1 Expression Using a Novel Multimodal Nanoparticle, Circulation Research 96:327-336.
Kelly, 2007, Miniaturizing chemistry and biology in microdroplets, Chem Commun 18:1773-1788.
Kerker, 1983, Elastic and inelastic light scattering in flow cytometry, Cytometry, 4:1-10.
Khandjian, 1986, UV crosslinking of RNA to nylon membrane enhances hybridization signals, Mol. Bio. Rep. 11: 107-115.
Kheir, 2012, Oxygen Gas-Filled Microparticles Provide Intravenous Oxygen Delivery, Science Translational Medicine 4 (140):140ra88 (10 pages).
Khodakov, 2016, Diagnostics based on nucleic acid sequence variant profiling: PCR, hybridization, and NGS approaches, Adv Drug Delivery Reviews 105:3-19.
Kim, 2003, Type II quantum dots: CdTe/CdSe (core/shell) and CdSe/ZnTe (core/shell) heterostructures, J. Am Chem Soc. 125:11466-11467.
Kim, 2004, Comparative study on sustained release of human growth hormone from semi-crystalline poly(L-lactic acid) and amorphous poly(D,L-lactic-co-glycolic acid) microspheres: morphological effect on protein release, Journal of Controlled Release, 98(1):115-125.
Kircher, 2010, High-throughput DNA sequencing-concepts and limitations, Bioessays 32(6):524-536.
Kiss et al., "High-Throughput Quantitative Polymerase Chain Reaction in Picoliter Droplets," Analytical Chemistry, December, vol. 80, No. 23, pp. 8975-8981. (Year: 2008). *
Kiss et al., "High-Throughput Quantitative Polymerase Chain Reaction in Picoliter Droplets," Analytical Chemistry, vol. 80, pp. 8975-8981. (Year: 2008). *
Kiss, 2008, High-throughput quantitative polymerase chain reaction in picoliter droplets, Anal. Chem 80:8975-8981.
Kitagawa, 1995, Manipulation of a single cell with microcapillary tubing based on its electrophoretic mobility, Electrophoresis 16:1364-1368.
Klug, 1994, All you wanted to know about selex, Molecular Biology Reports, 20:97-107.
Klug, 1995, Gene Regulatory Proteins and Their Interaction with DNA, Ann NY Acad Sci, 758: 143-60.
Klug, 1995, Protein motifs 5. Zinc fingers, FASEB J 9(8):597-604.
Knaak, 1995, Development of partition coefficients, Vmax and Km values, and allometric relationships, Toxicol Lett. 79 (1-3):87-98.
Knight, 1998, Hydrodynamic Focusing on a Silicon Chip: Mixing Nanoliters in Microseconds, Physical Review Lett 80 (17):3863-3866.
Koeller, 2001, Enzymes for chemical synthesis, Nature 409:232-240.
Kohara, 2002, DNA probes on beads arrayed in a capillary, ‘Beadarray’-exhibited high hybridization performance, 30, e87, pp. 1-7.
Kohler, 1975, Continuous cultures of fused cells secreting antibody of predefined specificity, Nature, 256:495-7.
Kojima, 2005, PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets. Nucleic Acids Res. 33:e150, 9 pages.
Kolb, 1995, Cotranslational folding of proteins, Biochem Cell Biol, 73:1217-20.
Komatsu, 2001, Roles of cytochromes P450 1A2, 2A6, and 2C8 in 5-fluorouracil formation rom tegafur, an anticancer prodrug, in human liver microsomes. Drug Met. Disp., 28:1457-1463.
Kopp, 1998, Chemical amplification: continuous flow PCR on a chip, Science, 280:1046-48.
Koster, 2008, Drop-based microfluidic devices for encapsulation of single cells, Lab on a Chip 8:1110-1115.
Kowalczykowski, 1994, Biochemistry of homologous recombination in Escherichia coli, Microbiol Rev 58(3):401-65.
Kozbor, 1984, A human hybrid myeloma for production of human monoclonal antibodies, J. Immunol., 133:3001-3005.
Krafft, 1991, Synthesis and preliminary data on the biocompatibility and emulsifying properties of perfluoroalkylated phosphoramidates as injectable surfactants, Eur. J. Med. Chem., 26:545-550.
Krafft, 2001, Fluorocarbons and fluorinated amphiphiles in drug delivery and biomedical research, Adv Rev Drug Disc 47:209-228.
Krafft, 2003, Emulsions and microemulsions with a fluorocarbon phase, Colloid and Interface Science 8(3):251-258.
Kralj, 2005, Surfactant-enhanced liquid-liquid extraction in microfluidic channels with inline electric-field enhanced coalescence, Lab Chip 5:531-535.
Krebber, C, et al., Selectivity-infective phage (SIP): a mechanistic dissection of a novel in vivo selection for protein-ligand interactions, Journal of Molecular Biology, 268, 607-618 (1997).
Kricka, 1996, Micromachining: a new direction for clinical analyzers, Pure and Applied Chemistry 68(10):1831-1836.
Kricka, 2003, Microchip PCR, Anal Bioanal Chem 377(5):820-825.
Kritikou, 2005, "It's cheaper in the Picolab," Nature, September, vol. 6, 1 page.
Kritikou, Ekat, "It's cheaper in the Picolab," Nature, September, vol. 6, p. 668. (Year: 2005).
Krumdiek, 1980, Solid-phase synthesis of pteroylpolyglutamates, Methods Enzymol, 524-29.
Kruth, 2003, Lasers and materials in selective laser sintering, Assembly Automation, 23(4):357-371.
Kuhlmann, 2009, Fixation of biological specimens, Division of Radiooncology, Germany, 12 pages.
Kumagai, 1994, Ablation of polymer films by a femtosecond high-peak-power Ti:sapphire laser at 798 nm, Applied Physics Letters, 65(14):1850-1852.
Kumar, 1989, Activity and kinetic characteristics of glutathione reductase in vitro in reverse micellar waterpool, Biochem Biophys Acta, 996(1-2):1-6.
Kumaresan, 2008, High-throughput single copy DNA amplification and cell analysis in engineered nanoliter droplets, Anal Chem, 80:3522-3529.
Lage, 2003, Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement amplification and array-CGH, Genome Res 13:294-307.
Laird, 2013, Rapid Quantification of the Latent Reservoir for HIV-1 Using a Viral Outgrowth Assay, PLOS Pathogens 9(5):e1003398.
Lamprecht, 2004, pH-sensitive microsphere delivery increases oral bioavailability of calcitonin, J Control Rel 98(1):1-9.
Lancet, 1993, Probability model for molecular recognition in biological receptor repertoirs, PNAS 90(8):3715-9.
Landergren, 1988, A ligase mediated gene detection technique, Science 241(4869):1077-80.
Langmuir, Directing Droplets Using Microstructured Surfaces, vol. 22 No. 14, Jun. 9, 2006 p. 6161-6167.
Lasheras, 1998, Breakup and atomization of a round water jet by a high speed annular air jet, J Fluid Mech 357:351-379.
Laufer, 1996, Introduction to Optics and Lasers in Engineering, Cambridge University Press, Cambridge UK:156-162.
Leamon, 2003, A massively parallel pictoterplate based platform for discrete picoliter-scale PCR, Electrophoresis 24:3769-3777.
Leary, 2000, Application of advanced cytometric and molecular technologies to minimal residual disease monitoring, Proc SPIE 3913:36-44.
Lee, 2000, Circulating flows inside a drop under time-periodic non-uniform electric fields, Phys Fuilds 12(8):1899-1910.
Lee, 2001, Preparation of silica particles encapsulating retinol using O/W/O multiple emulsions, J Coll Interface Sci 240 (1):83-89.
Lee, 2002, Effective formation of silicone-in-fluorocarbon-in-water double emulsions, J Disp Sci Tech 23(4):491-497.
Lee, 2002, Investigating the target recognition of DNA cytosine-5 methyltransferase Hhal by library selection using in vitro compartmentalisation (IVC), Nucleic Acids Res 30:4937-4944.
Lee, 2004, Special issue on biomedical applications for MEMS and microfluidics, Proc IEEE 92(1):3-5.
Lemof, 2003, An AC magnetohydrodynamic microfluidic switch for Micro Total Analysis Systems, Biomed Microdev 5 (I):55-60.
Leng 2009, Microfluidic crystalizaiton,Lab Chip 9:24-23.
Leng, 2010, Agarose droplet microfluidics for highly parallel and efficient single molecule emulsion PCR, Lab Chip 10:2841-2843.
Lesley, 1991, Use of in vitro protein synthesis from PCR-generated templates to study interaction of E coli transcription factors with core RNA polymerase, J Biol Chem 266(4):2632-8.
Lesley, 1995, Preparation and use of E. coli S-30 extracts, Methods Mol Biol 37:265-78.
Leung, 1989, A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction, Technique 1:11-15.
Li et al., 2003, Polydimethylsioxane 1-3 fluidic interconnects for microfluidic systems, IEEE Trans Adv Pack 36 (3):242-247.
Li, 1995, Single-step procedure for labeling DNA strand breaks with fllourescein—or BODIPY—conjugated deoxynucleotides, Cytometry 20:172-180.
Li, 1997, Transport, manipulation, and reaction of biological cells on-chip using electrokinetic effects, Anal Chem 69 (8):1564-1568.
Li, 2005, Multiplexed detection of pathogen DNA with DNA-based fluorescence nanobarcodes, Nat Biotech 23 (7):885-889.
Li, 2006, Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins, PNAS 103:19243-19248.
Li, 2018, Microfluidic fabrication of microparticles for biomedical applications, Chem Soc Rev 47(15):5646-5683.
Liao, 1986, Isolation of a thermostable enzyme variant by cloning and selection in a thermophile, PNAS 83:576-80.
Lim, 1980, Microencapsulated islets as bioartificial endocrine pancreas, Science 210(4472):908-10.
Lin et al., Self-Assembled Combinatorial Encoding Nanoarrays for Multiplexed Biosensing, Nanoletter, 2007, vol. No. 2 p. 507-512.
Lin, 2007, Self-assembled combinatorial encoding nanoarrays for multiplexed biosensing, Nano Lett 7(2):507-512.
Link, 2004, Geometrically mediated breakup of drops in microfluidic devices, Phys Rev Lettv92(5):054503-1-4.
Link, 2006, Electric control droplets in microfluidic devices, Angew Chem Int Ed 45:2556-2560.
Lipinski, 2001, Experimental and computational approaches to estimate solubility and permeability in drug discovery, , Adv Drug Deliv Rev 46:3-26.
Lipkin, 1988, Biomarkers of increased susceptibility to gastreointestinal cancer: new application to studies of cancer prevention in human subjects, Cancer Res 48:235-245.
Liu, 2000, Passive mixing in a three-dimensional serpentine microchannel, J MEMS 9(2):190-197.
Liu, 2002, Fabrication and characterization of hydrogel-based microvalves, Mecoelectromech. Syst.11:45-53.
Lizardi, 1998, Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat Genet 19(3):225-32.
Lo, 2007, Digital PCR for the molecular detection of fetal chromosomal aneuploidy, PNAS 104(32):13116-13121.
Loakes, 1994, 5-Nitroindole as a universal base analogue, Nucleic Acids Res 22:4039-4043.
Loakes, 1997, Stability and structure of DNA oligonucleotides containing non-specific base analogues, J Mol Biol 270:426-435.
Lodish, 2000, Structure of Nucleic Acids, Section 4.1 , Molecular Cell Biology, 4th edition, New York, 1-3.
Loeker et al., Colloids and Surfaces A: Physicochem. Eng. Aspects 214:143-150, (2003).
Loeker, 2003, FTIR analysis of water in supercritical carbon dioxide microemulsions using monofunctional perfluoropolyether surfanctants, Colloids and Surfaces A: Phys Eng Asp 214:143-150.
Loo, 2004, Nanoshell Enabled Photonics-Based Imaging and Therapy of Cancer, Technology in Cancer Research & Treatment 3(1):33-40.
Lopez-Herrera, 1995, The electrospraying of viscous and non-viscous semi-insulating liquids: scaling laws, Bull Am Phys Soc 40 (12):2041.
Lopez-Herrera, 1999, One-dimensional simulation of the breakup of capillary jets of conducting liquids application to EHD spraying, Aerosol Set 30(7):895-912.
Lopez-Herrera, 2003, Coaxial jets generated from electrified Taylor cones, Aerosol Sci 34:535-552.
Lorenceau, 2005, Generation of polymerosomes from double-emulsions, Langmuir 21(20):9183-9186.
Lorenz, 1991, Isolation and expression of a cDNA encoding Renilla reniformis luciferase, PNAS 88(10):4438-42.
Loscertales, 2002, Micro/nano encapsulation via electrified coaxial liquid jets, Science 295(5560):1695-1698.
Lowe, 2002, Perfluorochemical respiratory gas carriers: benefits to cell culture systems, J Fluorine Chem 118:19-26.
Lu, 2007, Robust fluorescein-doped silica nanoparticles via dense-liquid treatment, Colloids and Surfaces A Phys Eng Asp 303(3):207-210.
Luft, Detection of integrated papillomavirus sequences by ligation-mediaated PCR (DIPS-PCR) and molecular characterization in cervical cancer cells, In J Cancer 92:9-17 ,2001.
Luisi, 1987, Activity and conformation of enzymes in reverse micellar solutions, Meth Enzymol 136:188-216.
Lund, 1988, Assesment of methods for covalent binding of nucleic acids to magnetic beads, Dynabeads, and the characteristics of the bound nucleic acids in hybridization reactions, Nucleic Acids Res 16(22):10861-10880.
Lunderberg, 1995, Solid-phase technology: magnetic beads to improve nucleic acid detection and analysis, Biotech Ann Rev 1:373-401.
Lundstrom, 2002, Breakthrough in cancer therapy: Encapsulation of drugs and viruses, Curr Drug Disc 19-23.
Lyne, 2002, Structure-based virtual screening: an overview, Drug Disc Tod 7(20):1047-1055.
Ma, 1993, In vitro protein engineering using synthetic tRNA(Ala) with different anticodons, Biochemistry 32 (31):7939-45.
Machine translation of JP 2002-282682 (26 pages).
Mackenzie, 1985, IABS Symposium on Reduction of Animal Usage in the Development and Control of Biological Products, London, UK, 16 pages.
Mackenzie, 1986, The application of flow microfluorimetry to biomedical research and diagnosis: a review, Dev Biol Stand 64:181-193.
Maclean, 1999, Glossary of terms used in combinatorial chemistry, Pure Appl. Chem. 71(12):2349-2365.
Magdassi, 1984, Multiple Emulsions: HLB Shift Caused by Emulsifier Migration to External Interface, J. Colloid Interface Sci 97:374-379.
Mahajan, 1998, Bcl-2 and Bax Interactions in Mitochondria Probed with Green Florescent Protein and Fluorescence Resonance Energy Transfer, Nat. Biotechnol. 16(6): 547-552.
Mahjoob et al., Rapid microfluidic thermal cycler for polymerase chain reaction nucleic acid amplification. Int J HeatMass Transfer 2008;51:2109-22.
Mahjoob, 2008, Rapid microfluidic thermal cycler for polymerase chain reaction nucleic acid amplification. Int J HeatMass Transfer;51:2109-22.
Malmborg, A, et al., Selective phage infection mediated by epitope expression on F pilus, Journal of Molecular Biology, 273, 544-551 (1997).
Mammal Wikipedia.com accessed Sep. 22, 2011).
Manafi, 2000, New developments in chromogenic and fluorogenic culture media, 2000, International Journal of Food Microbiology, 60, 205-218.
Manley, 1983, In vitro transcription: whole cell extract, Methods Enzymol, 101:568-82.
Manz, 1991, Micromachining of monocrystalline silicon and glass for chemical analysis systems A look into next century's technology or just a fashionable craze, Trends in Analytical Chemistry 10(5):144-149.
Mao, 1991, Substrate effects on the enzymatic activity of alphachymotrypsin in reverse micelles, Biochem Biophys Res Commun, 178(3):1105-12.
Mao, 1992, Kinetic behaviour of alpha-chymotrypsin in reverse micelles: a stopped-flow study, Eur J Biochem 208 (1):165-70.
Mardis, 2008, The impact of next-generation sequencing technology on genetics, Trends Genet 24:133-141.
Margulies et al., 2005, Genome sequencing in microfabricated high-density picolitre reactors, Nature 437(7057).
Margulies, 2005, Genome sequencing in microfabricated high-density picolitre reactors, Nature 437(7057):376-380.
Marks, 1992, Bypassing immunization: building high affinity human antibodies by chain shuffling, BioTechnol 10:779-783.
Marques, 1996, Porous Flow within Concentric Cylinders, Bull Am Phys Soc Div Fluid Dyn 41:1768, 1 page.
Maruno, 1991, Fluorine containing optical adhesives for optical communications systems, J. Appl. Polymer. Sci. 42:2141-2148.
Mason, 1997, Shear Rupturing of Droplets in Complex Fluids, Langmuir, 13(17):4600-4613.
Mastrobattista, 2005, High-throughput screening of enzyme libraries: in vitro evolution of a beta-galactosidase by fluorescence-activated sorting of double emulsions, Chem. Biol. 12(12): 1291-1300.
Masui, 1998, Probing of DNA-Binding Sites of Escherichia coli RecA Protein Utilizing 1-anilinonaphthalene-8-Sulfonic Acid, Biochem 37(35):12133-12143.
Matayoshi, 1990, Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer, Science 247:954.
Matsubara, 2003, Detection of Single Nucleotide Substitution by Competitive Allele-Specific Short Oligonucleotide Hybridization (CASSOH) With Ummunochromatographic Strip, Human Mutation 22:166-172.
Mattheakis, 1994, An in vitro polysome display system for identifying ligands from very large peptide libraries, PNAS 91:9022-6.
Mayr, 2008, The Future of High-Throughput Screening, JBiomol Screen 13:443-448.
Mazutis, 2009, Droplet-Based Microfluidic Systems for High-Throughput Single DNA Molecule Isothermal Amplification and Analysis, Anal Chem 81(12):4813-4821.
Mazutis, 2009, Multi-step microfluidic droplet processing: kinetic analysis of an in vitro translated enzyme, Lab Chip 9:2902-2908.
McDonald, 2000, Fabrication of microfluidic systems in poly(dimethylsiloxane), Electrophoresis 21(1):27-40.
McDonald, 2002, Poly(dimethylsiloxane) as a material for fabricating microfluidic devices, Account Chem. Res. 35:491-499.
Melton, 1984, Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter, Nucl. Acids Res. 12(18):7035-7056.
Mendel, 1995, Site-Directed Mutagenesis with an Expanded Genetic Code, Annu Rev Biophys Biomol Struct, 24:435-62.
Mendieta, 1996, Complementary sequence correlations with applications to reflectometry studies, Instrumentation and Development 3(6):37-46.
Meng, 2015, Self-assembling amphiphilic poly(propargyl methacrylate) grafted DNA copolymers into multi-strand helices, Soft Matter 11(28):5610-5613.
Metzker, 2010, Sequencing Technologies—the next generation, Nature Reviews, vol. 11, pp. 31-46.
Meylan, 1995, Atom/fragment contribution method for estimating octanol-water partition coefficients, J Pharm Sci. 84 (1):83-92.
Michalatos-Beloin, 1996, Molecular haplotyping of genetic markers 10 kb apart by allele-specific long-range PCR, Nucleic Acids Research, 24:4841-4843.
Miele, 1983, Autocatalytic replication of a recombinant Rna, J Mol Biol, 171:281-95.
Milstein, 1983, Hybrid hybridomas and their use in immunohistochemistry, Nature 305:537-540.
Mindlin, 1936, A force at a point of a semi-infinite solid, Physics, 7:195-202.
Minshuil, 1999, Protein evolution by molecular breeding, Curr Opin Chem Biol 3(3): 284-90.
Miroux, 1996, Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels, J of Mol Biol 260(3):289-98.
Miyawaki, 1997, Fluorescent Indicators for Ca2+ Based on Green Fluorescent Proteins and Calmodulin, Nature, 388: 882-887.
Mize, 1989, Dual-enzyme cascade—an amplified method for the detection of alkaline phosphatase, Anal Biochem 179 (2): 229-35.
Mock, 1985, A fluorometric assay for the biotin-avidin interaction based on displacement of the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid, Anal Biochem, 151:178-81.
Moldavan, 1934, Photo-electric technique for the counting of microscopical cells, Science 80:188-189.
Monie, 2005, A Novel Assay Allows Genotyping of the Latent Reservoir for Human Immunodeficiency Virus Type 1 in the Resting CD4+ T Cells of Viremic Patients, Journal of Virology, 79(8):5185-5202.
Montigiani, 1996, Alanine substitutions in calmodulin-binding peptides result in unexpected affinity enhancement, J Mol Biol, 258:6-13.
Moore, 1995, Exploration by lamp light, Nature, 374:766-7.
Morrison, 1984, Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains, PNAS 81:6851-6855.
Moudrianakis, 1965, Base sequence determination in nucelic acids with the electron microscope 3. Chemistry and microscopy of guanine-labeled DNA, PNAS 53:564-71.
Mueth, 1996, Origin of stratification in creaming emulsions, Physical Review Letters 77(3):578-581.
Mulbry, 1989, Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein. J Bacteriol, 171: 6740-6746.
Mulder, 1993, Characterization of two human monoclonal antibodies reactive with HLA-B12 and HLA-B60, respectively, raised by in vitro secondary immunization of peripheral blood lymphocytes, Hum. Immunol 36(3):186-192.
Munson, 1980, Ligand: a versatile computerized approach for characterization of ligand-binding systems, Analytical Biochemistry, 107:220-239.
Murinae (Wikipedia.com accessed Mar. 18, 2013).
Nakano, 1994, High speed polymerase chain reaction in constant flow, Biosci Biotech and Biochem, 58:349-52.
Nakano, 2003, Single-molecule PCR using water-in-oil emulsion, J Biotech, 102:117-124.
Nakano, 2005, Single-molecule reverse transcription polymerase chain reaction using water-in-oil emulsion, J Biosci Bioeng 99:293-295.
Nametkin, 1992, Cell-free translation in reversed micelles, FEB Letters, 309(3):330-32.
Narang, 1979, Improved phosphotriester method for the synthesis of gene fragments, Methods Enzymol, 68:90-98.
Neiman, 2011, Decoding a substantial set of samples in parallel by massive sequencing, PLoS ONE 6(3):1-7.
Nelson, 1989, Bifunctional oligonucleotide probes synthesized using a novel CPG support are able to detect single base pair mutations, Nucl Acids Res 17(18): 7187-7194.
Nemoto, 1997, In vitro virus: bonding of mRNA bearing puromycin at the 3 terminal end to the C-terminal end of its encoded protein on the ribosome in vitro, Federation of European Biochemical Societies, 414:405-8.
Ness, 2000, Molecular Breeding: the natural approach to protein design. Adv Protein Chem, 55: 261-292.
Ng, 2003, Protein crystallization by capillary counter-diffusion for applied crystallographic structure determination, J. Struct. Biol, 142:218-231.
Ng, 2006, Factors affecting flow karyotype resolution, Cytometry, Part A 69A: 1028-1036.
Nguyen, 2006, Optical detection for droplet size control in microfluidic droplet-based analysis systems, Sensors and Actuators B 117(2):431-436.
Nihant, 1994, Polylactide Microparticles Prepared by Double Emulsion/Evaporation Technique. I. Effect of Primary Emulsion Stability, Pharmaceutical Research, 11(10):1479-1484.
Nisisako, 2002, Droplet formation in a microchannel network, Lab Chip 2:24-26.
Nisisako, 2002, Formation of droplets using branch channels in a microfluidic circuit, Proceedings of the SICE Annual Conference. International Session Papers 1262-1264.
Nisisako, 2005, Controlled formulation of monodisperse double emulsions in a multiple-phase microluidic system, Sot Matter, 1:23-27.
Nisisako, 2008, Microstructured Devices for Preparing Controlled Multiple Emulsions. Chem. Eng. Technol 31 (8):1091-1098.
Nof, 2002, Drug-releasing scaffolds fabricated from drug-loaded microspheres, J. Biomed Mater Res 59:349-356.
Norman, 1980, Flow Cytometry, Med. Phys., 7(6):609-615.
Notice of Refusal for Application No. 04782399.2 dated Apr. 10, 2013 (10 pages).
Nygren, 1982, Conjugation of horseradish peroxidase to Fab fragments with different homobifunctional and heterobifunctional cross-linking reagents. A comparative study, J. Histochem. and Cytochem. 30:407-412.
Obeid, 2003, Continuous-flow DNA and RNA amplification chip combined with laser-induced fluorescence detection, Anal Chimica Acta 494(1-2):1-9.
Oberholzer, 1995, Enzymatic RNA replication in self-reproducing vesicles: an approach to a minimal cell, Biochem Biophys Res Commun 207(1):250-7.
Oberholzer, 1995, Polymerase chain reaction in liposomes, Chem. Biol. 2(10):677-82.
Obukowicz, 1988, Secretion and export of IGF-1 in Escerichia coli strain JM101, Mol Gen Genet, 215:19-25.
Office Action for U.S. Appl. No. 11/360,845 Dated Nov. 19, 2013, 16 pages.
Office Action for U.S. Appl. No. 13/679,190 dated Dec. 2, 2013, 13 pages.
Office Action mailed Jun. 5, 2014 for U.S. Appl. No. 13/679,190.
Ogura, 1955, Catalase activity at high concentrations of hydrogen peroxide, Archs Biochem Biophys, 57: 288-300.
Oh et al., World-to-chip microfluidic interface with built-in valves for multichamber chip-based PCR assays, Lab Chip, 2005, 5, 845-850.
Oh, 2002, Distribution of Macropores in Silica Particles Prepared by Using Multiple Emulsions, Journal of Colloid and Interface Science, 254(1): 79-86.
Oh, 2005, World-to-chip microfluidic interface with built-in valves for multichamber chip-based PCR assays, Lab Chip, 5, 845-850.
Okuno, 2003, Recent Advances in Optical Switches Using Silica-based PLC Technology, NTT Technical Review 1 (7):20-30.
Okushima, 2004, Controlled production of monodisperse double emulsions by two-step droplet breakup in microfluidic devices, Langmuir 20(23): 9905-8.
Olsen, 2000, Function-based isolation of novel enzymes from a large library, Nat Bioteoltnol 13(10):1071-4.
Omburo, 1992, Characterization of the zinc binding site of bacterial phosphotriesterase, J of Biological Chem, 267:13278-83.
Original and translated Notice of Final Rejection dated Nov. 19, 2013 for Japanese Patent Application 2008-550290 (5 pages).
Original and translated Notice of Reasons for Rejection dated Apr. 10, 2013 for Japanese Patent Application 2008-550290 (6 pages).
Oroskar, 1996, Detection of immobilized amplicons by ELISA-like techniques, Clin. Chem. 42:1547-1555.
Ostermeier, 1999, A combinatorial approach to hybrid enzymes independent of DNA homology, Nat Biotechnol, 17 (12):1205-9.
Ott, 1967, Biological and medical research annual report, Los Alamos Scientific Laboratory, 14 pages.
Ouelette, 2003, A new wave of microfluidic devices, Indust Physicist pp. 14-17.
Pabit, 2002, Laminar-Flow Fluid Mixer for Fast Fluorescence Kinetics Studies, Biophys J 83:2872-2878.
Paddison, 2002, Stable suppression of gene expression by RNAi in mammalian cells, PNAS 99(3):1443-1448.
Pain, 1981, Preparation of protein A-peroxidase mono conjugate using a heterobifunctional reagent, and its use in enzyme immunoassays, J Immunol Methods, 40:219-30.
Pannacci, 2008, Equilibrium and Nonequilibrium States in Microluidic Double Emulsions Physical Review Leters, 101 (16):164502.
Park, 2001, Model of Formation of Monodispersed Colloids, J. Phys. Chem. B 105:11630-11635.
Park, 2003, Cylindrical compact thermal-cycling device for continuous-flow polymeras chain reaction, Anal Chem, ACS, 75:6029-33.
Parker, 2000, Development of high throughput screening assays using fluorescence polarization: nuclear receptor-ligand-binding and kinase/phosphatase assays, J Biomol Screen, 5(2): 77-88.
Pasternak, 2013, Cell-associated HIV RNA: a dynmic biomarker of viral persistence, Retrovirology 10:41.
Patel, 2003, Formation of Fluorinated Nonionic Surfactant Microemulsions in Hydrfuorocarbon 134a, Journal of Colloid and Interface Science, 258, 345-353.
Pedersen, 1998, A method for directed evolution and functional cloning of enzymes, PNAS 95(18):10523-8.
Pekin, 2011, Quantitative and sensitive detection of rare mutations using droplet-based microfluidics, Lab on a Chip 11 (13):2156-2166.
Pelham, 1976, An efficient mRNA-dependent translation system from reticulocyte lysates, Eur J Biochem 67:247-56.
Pelletier, 1999, An in vivo library-versus-library selection of optimized protein-protein interactions, Nature Biotechnology, 17:683-90.
Peng, 1998, Controlled Production of Emulsions Using a Crossflow Membrane, Particle & Particle Systems Characterization 15:21-25.
Pepe, 2004, Limitations of the odds ratio in gauging the performance of a diagnostic, prognostic, or screening marker, American Journal of Epidemiology 159(9):882-890.
Perelson, 1979, Theorectical studies of clonal selection: minimal antibody repertoire size and relaibility of self-non-self discrimination. J Theor Biol 81(4):645-70.
Perez-Gilabert, 1992, Application of active-phase plot to the kinetic analysis of lipoxygenase in reverse micelles, Biochemistry J. 288:1011-1015.
Petrounia, 2000, Designed evolution of enzymatic properties, Curr Opin Biotechnol, 11:325-330.
Pirrung, 1996, A General Method for the Spatially Defined Immobilization of Biomolecules on Glass Surfaces Using Caged Biotin, Bioconjug Chem 7: 317-321.
Plant (Wikipedia.com accessed Mar. 8, 2013).
Ploem, 1993, in Fluorescent and Luminescent Probes for Biological Activity Mason, T. G. Ed., Academic Press, Landon, pp. 1-11.
Pluckthun, 2000, In vitro selection and evolution of proteins, Adv Protein Chem, 55: 367-403.
Pollack, 1986, Selective chemical catalysis by an antibody, Science 234(4783):1570-3.
Pollack, 2002, Electrowetting-based actuation of droplets for integrated microfluidics, Lab Chip 2:96-101.
Pons, 2009, Synthesis of Near-Infrared-Emitting, Water-Soluble CdTeSe/CdZnS Core/Shell Quantum Dots, Chemistry of Materials 21(8):1418-1424.
Poon, 2002, Differential DNA Methylation between Fetus and Mother as a Strategy for Detecting Fetal DNA in Maternal Plasma, Clinical Chemistry, 48:35-41.
Posner, 1996, Engineering specificity for folate into dihydrofolate reductase from Escherichia coli, Biochemistry, 35: 1653-63.
Priest, 2006, Generation of Monodisperse Gel Emulsions in a Microfluidic Device, Applied Physics Letters, 88:024106, 3 pages.
Puntambekar et al., 2002, Self aligning microfluidic interconnects for glass- and plastic-based microfluidic systems, J Micromech Microeng 12(1):35-40.
Qi, 1998, Acid Beta-Glucosidase: Intrinsic Fluorescence and Conformational Changes Induced by Phospholipids and Saposin C, Biochem., 37(33): 11544-11554.
Raghuraman, 1994, Emulston Liquid Membranes for Wastewater Treatment: Equilibrium Models for Some Typical Metal-Extractant Systems,Environ. Sci. Technol 28:1090-1098.
Ralhan, 2008, Discovery and Verification of Head-and-neck Cancer Biomarkers by Differential Protein Expression Analysis Using iTRAQ Labeling, Multidimensional Liquid Chromatography, and Tandem Mass Spectrometry, Mol Cell Proteomics 7(6):1162-1173.
Ramanan, 2016, Algae-bacteria interactions, Biotech ADv 34:14-29.
Ramsey, 1999, The burgeoning power of the shrinking laboratory, Nat Biotechnol 17(11):1061-2.
Ramstrom, 2002, Drug discovery by dynamic combinatorial libraries, Nat Rev Drug Discov 1:26-36.
Rasmussen, 2013, Comparison of HDAC inhibitors in clinical development, Human Vacc Immunother 9(5):993-1001.
Raushel, 2000, Phosphotriesterase: an enzyme in search of its natural substrate, Adv Enzymol Relat Areas Mol Biol, 74: 51-93.
Rech, 1990, Introduction of a yeast artificial chromosome vector into Sarrachomyeces cervesia by electroporation, Nucleic Acids Res 18:1313.
Reyes, 2002, Micro Total Analysis Systems. 1. Introduction, Theory and Technology, Anal Chem 74(12):2623-2636.
Riechmann, 1988, Reshaping human antibodies for therapy, Nature, 332:323-327.
Riess, 2002, Fluorous micro- and nanophases with a biomedical perspective, Tetrahedron 58(20):4113-4131.
Roach, 2005, Controlling nonspecific protein adsorption in a plug-based microfluidic system by controlling inteifacial chemistry using fluorous-phase surfactants, Anal. Chem. 77:785-796.
Roberts, 1969, Termination factor for RNA synthesis, Nature, 224: 1168-74.
Roberts, 1975, Simian virus 40 DNA directs synthesis of authentic viral polypeptides in a linked transcription-translation cell-free system 72(5):1922-1926.
Roberts, 1997, RNA-peptide fusion for the in vitro selection of peptides and proteins, PNAS 94:12297-302.
Roberts, 1999, In vitro selection of nucleic acids and proteins: What are we learning, Curr Opin Struct Biol 9(4): 521-9.
Roberts, 1999, Totally in vitro protein selection using mRNA-protein fusions and ribosome display. Curr Opin Chem Biol 3(3), 268-73.
Roche, 2011, 454 Sequencing System Guidelines for Amplicon Experimental Design, 50 pages.
Rodriguez-Antona, 2000, Quantitative RT-PCR measurement of human cytochrome P-450s: application to drug induction studies. Arch. Biochem. Biophys., 376:109-116.
Rogers, 2005, Closing bacterial genoimc sequence gaps with adaptor-PCR, BioTechniques 39(1):1-3.
Rolland, 1985, Fluorescence Polarization Assay by Flow Cytometry, J. Immunol. Meth., 76(1): 1-10.
Rosenberg et al., Termination of transcription in bacteriophage lambda, J Biol Chem, 250: 4755-64 (1975).
Rosenberg, 1975, Inhibition of Human Factor IX by Human Antithrombin, J Biol Chem, 250: 4755-64.
Rosenberry, 1975, Acetylcholinesterase, Adv Enzymol Relat Areas Mol Biol, 43: 103-218.
Rotman, 1961, Measurement of activities of single molecules of beta-galactosidase, PNAS, 47:1981-91.
Rouzioux, 2013, How to best measure HIV reservoirs, Curr Op HIV AIDS 8(3):170-175.
Russon et al., Single-nucleotide polymorphism analysis by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device, Electrophoresis, 24:158-61 (2003).
Saarela et al., 2006, Re-useable multi-inlet PDMS fluidic connector, Sens Act B 114(1):552-557.
Saarela, 2006, Re-usable multi-inlet PDMS fluidic connector, Sensors Actuators B 114(1):552-57.
Sadtler, 1996, Achieving stable, reverse water-in-fluorocarbon emulsions, Angew Chem Int Ed 35(17):1976-1978.
Sadtler, 1999, Reverse water-In-fluorocarbon emulsions as a drug delivery system: an in vitro study, Colloids & Surfaces A: Phys Eng Asp 147:309-315.
Saiki, 1988, Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase, Science 239 (4839):487-91.
Sakamoto, 2005, Rapid and simple quantification of bacterial cells by using a microfluidic device, Appl Env Microb 71:2.
Salomon, 2019, Droplet-based single cell RNAseq tools: a practical guide, Lab on a Chip 19:1706-1727.
Sano, 1992, Immuno-PCR: very sensitive antigen-detection by means of sepcific Ab-DNA conjugates, Science 258 (5079):120-122.
SantaLucia, 1998, A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics, PNAS 95(4):1460-5.
Santos, 2007, Detection of Amyloid-b Oligomers in Human Cerebrospinal Fluid by Flow Cytometry and Fluorescence Resonance Energy Transfer, J of Alzheimer's Disease, 11:117-125.
Santra, 2006, Fluorescence lifetime measurements to determine the core-shell nanostructure of FITC-doped silica nanoparticles, J Luminescence 117(1):75-82.
Sawada, 1996, Synthesis and surfactant properties of novel fluoroalkylated amphiphilic oligomers, Chem Commun 2:179-190.
Schatz, 1996, Screening of peptide libraries linked to lac repressor, Meth Enzymol 267:171-91.
Schneegass, 2001, Miniaturized flow-through PCR with different template types in a silicone chip thermocycler, Lab on a Chip 1:42-9.
Schopman, 2012, Selective packaging of cellular miRNAs in HIV-1 particles, Virus Res 169(2):438-47.
Schubert, 2002, Designer Capsules, Nat Med 8:1362.
Schweitzer, 2000, Immunoassays with rolling circle DNA amplification, PNAS 97(18):10113-10119.
Schweitzer, 2001, Combining nucleic acid amplification and detection. Curr Opin Biotechnol 12(1):21-7.
Scott, 1948, The solubility of fluorocarbons, J Am Chem Soc 70:4090-4093.
Sedlak, 2013, Viral diagnostics in the era of digital polymerase chain reaction, Diag Microb Inf Dis 75(1):1-4.
Seethala, 1997, Homogeneous fluorescence polarization assay for Src-Family tyrosine kinases, Anal Biochem 253 (2):210-218.
Seiler, 1993, Planar glass chips for capillary electrophoresis: repetitive sample injection, quantitation, and separation efficiency, Anal Chem 65(10):1481-1488.
Selwyn, 1965, A simple test for inactivation of an enzyme during assay, Biochim Biophys Acta 105:193-195.
Sen, 2012, Development and validation of a new adenosine-independent index of stenosis severity from coronary wave-intensity analysis, Journal of the American College of Cardiology 59(15):1392-1402.
Seo, 2007, Microfluidic consecutive flow-focusing droplet generators, Soft Matter 3:986-992.
Seong, 2002, Efficient mixing and reactions within microfluidic channels using microbead-supported catalysts, J Am Chem Soc 124(45):13360-1.
Seong, 2002, Fabrication of microchambers defined by photopolymerized hydrogels and weirs within microfluidic systems, Anal Chem 74(14):3372-3377.
Sepp, 2002, Microbead display by in vitro compartmentalisation: selection for binding using flow cytometry, FEBS Letters 532:455-58.
Serpersu, 1985, Reversible and irreversible modification of erythrocyte membrane permeability by electric field, Biochim Biophys Acta 812(3):779-785.
Shapiro, 1983, Multistation multiparameter flow cytometry: a critical review and rationale, Cytometry 3: 227-243.
Shastry, 2006, Directing droplets using microstructured surfaces, Langmuir 22:6161-6167.
Shen, 2006, Eigengene-based linear discriminant model for tumor classification using gene expression microarray data, Bioinformatics 22(21):2635-2642.
Shendure, 2008, Next-generation DNA sequencing, Nature Biotechnology, 26(10):1135-1145.
Shestopalov, 2004, Multi-step synthesis of nanoparticles performed on millisecond time scale in a microfluidic droplet-based system, Royal Soc Chem 4:316-321.
Shim, Jung-uk, et al., Using Microfluidics to Decoupled Nucleation and Growth of Protein Crystals, Cryst. Growth, Des. 2007; 7(11): 2192-2194.
Shimizu, 1995, Encapsulation of biologically active proteins in a multiple emulsion, Biosci Biotech Biochem 59 (3):492-496.
Shtern, 1996, Hysteresis in swirling jets, J Fluid Mech 309:1-44.
Sia, 2003, Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies, Electrophoresis 24 (21):3563-3576.
Siemering, 1996, Mutations that suppress the thermosensitivity of green fluorescent protein, Curr Biol 6:1653-1663.
Silva-Cunha, 1998, W/O/W multiple emulsions of insulin containing a protease inhibitor and an absorption enhancer: biological activity after oral administration to normal and diabetic rats, Int J Pharm 169:33-44.
Sims, 2000, Immunopolymerase chain reaction using real-time polymerase chain reaction for detection, Anal. Biochem. 281(2):230-2.
Sista, 2007, Development of a Digital Microfluidic Lab-on-a-Chip for Automated Immunoassay with Magnetically Responsive Beads, Doctoral Thesis, Florida State University, 128 pages.
Sista, 2008, Development of a digital microfluidic platform for point care testing, Lab on a Chip 8:2091-2104.
Siwy, 2003, Electro-responsive asymmetric nanopores in polyimide with stable ion-current signal, Appl Phys A: Mat Sci Proc 76:781-785.
Slappendel, 1994, Normal cations and abnormal membrane lipids in the red blood cells of dogs with familial stomatocytosis hypertrophic gastritis, Blood 84:904-909.
Slob, 1997, Structural identifiability of PBPK models: practical consequences for modeling strategies and study designs, Crit Rev Toxicol. 27(3):261-72.
Smith, 1985, The synthesis of oligonucleotides containing an aliphatic amino group at the 5′ terminus: synthesis of fluorescent DNA primers for use in DNA sequence analysis, Nucl Acid Res 13:2399-2412.
Smith, 1986, Fluorescence detection in automated DNA sequence analysis, Nature 321:674-679.
Smith, 1989, Absolute displacement measurements using modulation of the spectrum of white light in a Michelson interferometer, Applied Optics, 28(16):3339-3342.
Smith, 1992, Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads, Science 258(5085):1122-1126.
Smith, 2010, Highly-multiplexed barcode sequencing: an efficient method for parallel analysis of pooled samples, Nucleic Acids Res 38(13):e142.
Smyth, 2000, Markers of apoptosis: methods for elucidating the mechanism of apoptotic cell death from the nervous system, Biotechniques 32:648-665.
Sohn, 2000, Capacitance cytometry: Measuring biological cells one by one, PNAS 97(20):10687-10690.
Sola, 2014, Fabrication of a microfluidic cell made of thiolene for microarray applications, 18th Int Conf Miniaturized Systems for Chem and Life Sciences, MicroTAS, San Antonio, TX 1719-1721.
Somasundaram, 1999, Gain studies of Rhodamine 6G dye doped polymer laser, J Photochem Photobiol 125 (1-3):93-98.
Song, 2002, Experimental test of scaling of mixing by chaotic advection in droplets moving through microfluidic channels, App Phy Lett 83(22):4664-4666.
Song, 2003, A microfluidic system for controlling reaction networks in time, Angew Chem Int Ed 42(7):768-772.
Song, 2003, Millisecond kinetics on a microluidic chip using nanoliters of reagents, J Am Chem Soc 125:14613-14619.
Song, 2006, Reactions in droplets in microfluidic channels, Angew chem Int ed 45(44):7336-7356.
Soni, 2007, Progress toward ultrafast DNA sequencing using solid-state nanopores, Clin Chem 53:1996-2001.
Soumillion, 2001, Novel concepts for the selection of catalytic activity. Curr Op Biotech 12:387-394.
Spiro, 2000, A bead-based method for multiplexed identification and quantitation of DNA sequences using flow cytometry, Appl Env Micro 66:4258-4265.
Sproat, 1987, The synthesis of protected 5′-mercapto-2′,5′-dideoxyribonucleoside-3′-0-phosphorainidites, uses of 5′-mercapto-oligodeoxyribonucleotides, Nucleic Acids Res 15:4837-4848.
Squires, 2005, Microfluidics: fluid physics at the nanoliter scale, Rev Mod Phys 77:977-1026.
Stauber, 1993, Rapid generation of monoclonal antibody-secreting hybridomas against African horse sickness virus by in vitro immunization and the fusion/cloning technique, J Immunol Meth 161(2):157-168.
Stemmer, 1994, DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. PNAS 91(22):10747-51.
Stemmer, 1994, Rapid evolution of a protein in vitro by DNA shuffling, Nature 370(6488):389-91.
Stober, 1998, Controlled growth of monodisperse silica spheres in the micron size range, J Colloid Interface Sci 26 (1):62-69.
Stofko, 1992, A single step purification for recombinant proteins, Febs Lett 302:274-278.
Stone, 2004, Engineering flows in small devices: microfluidics toward a lab-on-a-chip, Ann Rev Fluid Mech 36:381-441.
Strizhkov, 2000, PCR amplification on a microarray of gel-immobilized oligonucleotides: Detection of bacterial toxin- and drug-resistant genes and their mutations, BioTechniques 29(4):844-857.
Strommenger, 2003, Multiplex PCR assay for simultaneous detection of nine clinicly relevant antibiotic resistance genes in S aureus, J Clin Microb 41(9):4089-4094.
Stroock, 2002, Chaotic mixer for microchannels, Science 295(5555):647-651.
Studer, 1997, Fluorous synthesis: a fluorous-phase strategy for improving separation efficiency in organic synthesis, Science 275:823-826.
Sugiura, 2001, Interfacial tension driven monodispersed droplet formation from mtcrofabricated channel array, Langmuir 17:5562-5566.
Sugiura, 2002, Effect of channel structure on microchannel emuisification, Langmuir 18:5708-5712.
Sundberg, 1995, Spatially-addressable immobilisation of macromolecules on solid supports, J Am Chem Soc 117:12050-12057.
Sung, 2005, Chip-based microfluidic devices coupled with electrospray ionization-mass spectrometry, Electrophoresis 26:1783-1791.
Supplementary European Search Report and European Search Opinion, mailed Aug. 17, 2016, for EP 12745236.5 (5 pages).
Supplementary European Search Report and European Search Opinion, mailed Feb. 15, 2017, for EP 14784827.9 (8 pages).
Supplementary European Search Report and European Search Opinion, mailed Oct. 17, 2016, for EP 14778430.0 (8 pages).
Supplementary European Search Report and European Search Opinion, mailed Sep. 20, 2018, for EP 18157363.5 (8 pages).
Sutcliffe, 1986, Dynamics of UV laser ablation of organic polymer surfaces, J Appl Phys 60(9):3315-3322.
Suzuki, 1996, Random mutagenesis of thermus aquaticus DNA polmerase I: concordance of immutable sites in vivo with the crystal structure, PNAS 93:96701-9675.
Suzuki, 2013, A novel guidewire approach for handling acute-angle bifurcations, J Inv Cardiol 25(1):48-54.
Syed, 2009, Next-generation sequencing library preparation: simultaneous fragmentation and tagging using in vitro transposition, Nat Meth 6:1-2.
Takayama, 1999, Patterning cells and their environmnets using multiple laminar fluid flows in cappillary networks, PNAS 96:5545-5548.
Takeuchi, 2005, An axisymmetric flow-focusing microfluidic device, Adv Mater 17(8):1067-1072.
Taly, 2007, Droplets as microreactors for high-throughput biology, Chembiochem 8(3):263-272.
Tan, 2003, Controlled fission of droplet emulsions in bifurcating microfluidic channels, 12th Int Conf SSAM 28-31.
Tan, 2003, Microfluidic liposome generation from monodisperse droplet emulsion, Summer Bioeng Conf, Florida, 2 pages.
Tan, 2003, Monodisperse droplet emulsions in co-flow microfluidic channels, Micro TAS, 2 pages.
Tan, 2004, Design of microluidic channel geometries for the control of droplet volume, chemical concentration, and sorting, Lab Chip 4(4):292-298.
Tang, 2009, A multi-color fast-switching microfluidic droplet dye laser, Lab Chip 9:2767-2771.
Taniguchi, 2002, Chemical reactions in microdroplets by electrostatic manipulation of droplets in liquid media, Lab Chip 2:19-23.
Tawfik, 1998, Man-made cell-like compartments for molecular evolution, Nat Biotech 7(16):652-56.
Taylor, 1934, The formation of emulsions in definable field of flow, Proc R Soc London A 146(858):501-523.
Taylor, 1991, Characterization of chemisorbed monolayers by surface potential measurments, J Phys D Appl Phys 24:1443.
Tencza, 2000, Development of a fluorescence polarization-based diagnostic assay for equine infectious anemia virus, J Clin Microbiol 38(5):1854-185.
Terray, 2002, Fabrication of linear colloidal structures for microfluidic applications, Applied Phys Lett 81(9):1555-1557.
Terray, 2002, Microfluidic control using colloidal devices, Science 296(5574):1841-1844.
Tewhey et al., Microdroplet-based PCR enrichment for large scale targeted sequencing, Nature Biotechnology, 2009, vol. 27 (11) p. 1025-1031.
Tewhey, 2009, Microdroplet based PCR environment for large scale targeted sequence, Nat Biotech 27(11):1025-1031.
Theberge, 2010, Microdroplets in microfluidics: an evolving platform for discoveries in chemistry and biology, Angew Chem Int Ed 49(34):5846-5868.
Thompson, 1983, Introduction to Lithography, ACS Symp Ser 219:1-13.
Thorsen, 2001, Dynamic pattern formation in a vesicle-generating microfluidic device, Phys Rev Lett 86(18):4163-4166.
Thorsen, 2002, Microfluidic large-scale integration, Science 298:580-584.
Thorsen, 2003, Microfluidic technologies for highthroughput screening applications, California Institute of Technology.
Tice, 2003, Formation of droplets and mixing in multiphase microfluidics at low values of the reynolds and the capillary numbers, Langmuir 19:9127-9133.
Tice, 2004, Effects of viscosity on droplet formation and mixing in microfluidic channels, Analytica Chimica Acta 507:73-77.
Titomanlio, 1990, Capillary experiments of flow induced crystallization of HDPE, AlChe J 36(1):13-18.
Tleugabulova, 2004, Evaluating formation and growth mechanisms of silica particles using fluorescence anisotropy decay analysis, Langmuir 20(14):5924-5932.
Tokatlidis, 1995, Nascent chains: folding and chaperone intraction during elongation on ribosomes, Philos Trans R Soc Lond B Biol Sci, 348:89-95.
Tokeshi, 2002, Continuous-flow chemical processing on a microchip by combining microunit operations and a multiphase flow network, Anal Chem 74(7):1565-1571.
Tokumitsu, 1999, Preparation of gadopentetic acid-loaded chitosan microparticles for gadolinium neutron-capture therapy of cancer by a novel emulsion-droplet coalescence technique, Chem Pharm Bull 47(6):838-842.
Tonelli et al., 2002, Perfluoropolyether functional oligomers: unusual reactivity in organic chemistry, Journal of fluorine Chemistry, 118; 107-121.
Trolier-McKinstry, 2004, Thin Film Piezoelectric for MEMS, Journal of Electroceramics 12:7-17.
Tsuchiya, 2007, On-chip polymerase chain reaction microdevice employing a magnetic droplet-manipulation system, Sens Actuators B 130:583-588.
Tuzel, 2006, Region Covariance: A Fast Descriptor for Detection and Classification, European Conference on Computer Vision (ECCV), 14 pages.
Umbanhowar, 2000, Monodisperse Emulsion Generation via Drop Break Off in a Coflowing Stream, Langmuir 16 (2):347-351.
Unger, 2000, Monolithic microfabricated valves and pumps by multylayer soft lithography, Science 288(5463):113-116.
Utada et al., 2005, Monodisperse Double Emulsions Generated from a Microcapillary Device, Science 308:537-541.
Utada, 2005, Monodisperse double emulsions generated from a microcapillary device, Science, 308:537-541.
Vainshtein, 1996, Peptide rescue of an N-terminal truncation of the stoffel fragment of Taq DNA polymerase, Protein Science, 5:1785-92.
Van der Sluis, 2013, Dendritic Cell-induced Activation of Latent HIV-1 Provirus in Actively Proliferating Primary T Lymphocytes, PLOS Pathog. 9(3): 16 pages.
Van Dilla, 1968, The fluorescent cell photometer: a new method for the rapid measurement of biological cells stained with fluorescent dyes, Annual Report of the Los Alamos Scientific Laboratory of the University of California (Los Alamos, NM), Biological and Medical Research Groupp (H-4) of the Health Division, Compiled by D. G. Ott, pp. 100-105.
Van Dilla, 1969, Cell Microfluorometry: A Method for Rapid Fluorescence Measurement, Science 163(3872):1213-1214.
Vanhooke, 1996, Three-dimensional structure of the zinc-containing phosphotrieesterase with the bound substrate analog diethy 4-methylbenzylphosphonate, Biochemistry 35:6020-6025.
Varga, 1991, Mechanism of allergic cross-reactions-I. Multispecific binding of ligands to a mouse monoclonal anti-DNP IgE antibody. Mol Immunol 28(6), 641-54.
Vary, 1987, A homogeneous nucleic acid hybridization assay based on strand displacement, Nucl Acids Res 15 (17):6883-6897.
Venkateswaran, 1992, Production of Anti-Fibroblast Growth Factor Receptor Monoclonal Antibodies by In Vitro Immunization, Hybirdoma, 11(6):729-739.
Verhoeyen, 1988, Reshaping human antibodies: grafting an antilysozyme activity, Science, 239:1534-1536.
Viruses ( Wikipedia.com, accessed Nov. 24, 2012).
Vogelstein, 1999, Digital PCR, PNAS 96(16):9236-9241.
Voss, 1993, Kinetic measurements of molecular interactions by spectrofluorometry, J Mol Recognit, 6:51-58.
Wahler, 2001, Novel methods for biocatalyst screening. Curr Opin Chem Biol, 5: 152-158.
Walde, 1988, Structure and activity of trypsin in reverse micelles, Eur J Biochem, 173(2):401-9.
Walde, 1993, Spectroscopic and kinetic studies of lipases solubilized in reverse micelles, Biochemistry, 32(15):4029-34.
Walde, 1994, Oparin's reactions revisited: enzymatic synthesis of poly(adenylic acid) in micelles and self-reproducing vesicles. J Am Chem Soc, 116: 7541-7547.
Walker, 1992, Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system, PNAS 89 (1):392-6.
Walker, 1992, Strand displacement amplification—an isothermal, in vitro DNA amplification technique, Nucleic Acid Res, 20(7):1691-6.
Wang, 1989, Quantitation of mRNA by the polymerase chain reaction. Proc natl Acad Sci USA 86(24), 9717-21.
Wang, 1990, Design and synthesis of new fluorogenic HIV protease substrates based on resonance energy transfer, Tetrahedron Lett., 31:6493.
Wang, 2002, Preparation of Titania Particles Utilizing the Insoluble Phase Interface in a MicroChannel Reactor, Chemical Communications 14:1462-1463.
Wang, 2008, DEP actuated nanoliter droplet dispensing using feedback control, Lab on a Chip 9:901-909.
Wang, 2010, Quantifying EGFR Alterations in the Lung Cancer Genome with Nanofluidic Digital PCR Arrays, Clinical Chemistry 56:4.
Wang, Jun, et al., Quantifying EGFR Alterations in the Lung Cancer Genome with Nanofluidic Digital PCR Arrays, Clinical Chemistry 56:4 (2010).
Warburton, 1993, Microcapsules for Multiple Emulsions, Encapsulation and Controlled Release, Spec Publ R Soc Chem, 35-51.
Wasserman, 1989, Structure and reactivity of allyl-siloxane monolayers formed by reaction of allcyltrichlorosilanes on silicon substrates, Langmuir 5:1074-1087.
Weaver, Suzanne, et al., Taking qPCR to a higher level: Analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution, Methods 50, 271-276 (2010).
Weil, 1979, Selective and accurate initiation of transcription at the Ad2 major late promotor in a soluble system dependent on purified RNA polymerase II and DNA, Cell, 18(2):469-84.
Werle, 1994, Convenient single-step, one tube purification of PCR products for direct sequencing, Nucl Acids Res 22 (20):4354-4355.
Wetmur, 2005, Molecular haplotyping by linking emulsion PCR: analysis of paraoxonase 1 haplotypes and phenotypes, Nucleic Acids Res 33(8):2615-2619.
White, 2009, Digital PCR provides sensitive and absolute calibration for high throughput sequencing, BMC Genomics 10:116.
Wick, 1996, Enzyme-containing liposomes can endogenously produce membrane-constituting lipids, Chem Biol 3 (4):277-85.
Wiggins, 2004, Foundations of chaotic mixing, Philos Transact A Math Phys Eng Sci 362(1818):937-70.
Williams, 1979, Methotrexate, a high-affinity pseudosubstrate of dihydrofolate reductase, Biochemistry, 18(12):2567-73.
Williams, 2006, Amplification of complex gene libraries by emulsion PCR, Nature Methods 3(7):545-550.
Wilson, 1999, In vitro selection of functional nucleic acids, Ann. Rev. Biochem. 68: 611-647.
Wittrup, 2001, Protein engineering by cell-surface display. Curr Opin Biotechnology, 12: 395-399.
Wittwer, C.T., et al., Automated polymerase chain reaction in capillary tubes with hot air, Nucleic Acids Res., 17(11) 4353-4357 (1989).
Wittwer, Carl T., et al., Minimizing the Time Required for DNA Amplification by Efficient Heat Transfer to Small Samples, Anal. Biochem., 186, 328-331 (1990).
Wolff, 2003, Integrating advanced functionality in a microfabricated high-throughput fluorescent-activated cell sorter, Lab Chip, 3(1): 22-27.
Woolley, Adam T. and Mathies, Richard A., Ultra-high-speed DNA fragment separations using microfabricated capillary array electrophoresis chips, Proc. Natl. Acad. Sci. USA, 91, 11348-11352 (Nov. 1994).
Woolley, Adam T., et al., Functional Integration of PCR Amplification and Capillary Electrophoresis in a Microfabricated DNA Analysis Device, Anal. Chem. 68, 4081-4086 (Dec. 1, 1996).
Written Opinion for PCT/US2004/027912 dated Jan. 26, 2005, 6 pages.
Writtion Opinionfor PCT/US2006/001938 dated May 31, 2006, 8 pages.
Wronski, 2002, Two-color, fluorescence-based microplate assay for apoptosis detection. Biotechniques, 32:666-668.
Wu, 1989, The ligation amplification reaction (LAR)-amplification of specific DNA sequences using sequential rounds of template-dependent ligation, Genomics 4(4):560-9.
Wyatt, 1991, Synthesis and purification of large amounts of RNA oligonucleotides, Biotechniques 11(6):764-9.
Xia and Whitesides, Soft Lithography, Angew. Chem. Int. Ed. 37:550-575 (1998).
Xia, 1998, Soft Lithography, Ann. Rev. Mat. Sci. 28:153-184.
Xiao, 2007, Rapid DNA mapping by fluorescent single molecule detection, Nucleic Acids Research 35:1-12.
Xing, 2011, Novel structurally related compounds reactivate latent HIV-1 in a bcl-2-transduced primary CD4+ T cell model without inducing global T cell activation, Journal of Antimicrobial Chemotherapy, 67(2):398-403.
Xu et al., Design of 240, 000 orthogonal 25mer DNA barcode probes, PNAS, Feb. 17, 2009, 106(7) p. 2289-2294.
Xu, 2005, Generation of monodisperse particles by using microfluidics: control over size, shape, and composition, Angew. Chem. Int. Ed. 44:724-728.
Yamagishi, 1990, Mutational analysis of structure-activity relationships in human tumor necrosis factor-alpha, Protein Eng, 3:713-9.
Yamaguchi, 2002, Insulin-loaded biodegradable PLGA microcapsules: initial burst release controlled by hydrophilic additives, Journal of Controlled Release, 81(3): 235-249.
Yelamos, 1995, Targeting of non-Ig sequences in place of the V segment by somatic hypermutation. Nature 376 (6537):225-9.
Yershov, 1996, DNA analysis and diagnostics on oligonucleotide microchips, PNAS 93(10):4913-4918.
Yonezawa, 2003, DNA display for in vitro selection of diverse peptide libraries, Nucleic Acids Research, 31(19): e118, 5 pages.
Yu, 1997, Specific inhibition of PCR by non-extendable oligonucleotides using a 5′ to 3′ exonuclease-deficient DNA polymerase, Biotechniques 23(4):714-6, 718-20.
Yu, 2001, Responsive biomimetic hydrogel valve for microfluidics. Appl. Phys. Lett 78:2589-2591.
Yu, 2002, Environmental Carcinogenic Polycyclic Aromatic Hydrocarbons: Photochemisrty and Phototoxicity, J Environ Scie Health C Environ Carcinog Exotoxicol Rev, 20(2), 1-43.
Yu, 2007, Quantum dot and silica nanoparticle doped polymer optical fibers, Optics Express 15(16):9989-9994.
Zaccolo, 1996, An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues. J Mol Biol 255(4):589-603.
Zakrzewski, 1980, Preparation of tritiated dihydrofolic acid of high specific activity, Methods Enzymol, 529-533.
Zaug, 1986, The intervening sequence RNA of Tetrahymena is an enzyme, Science 231(4737):470-5.
Zaug, 1986, The Tetrahymena intervening sequence ribonucleic acid enzyme is a phosphotransferase and an acid phosphatase, Biochemistry 25(16):4478-82.
Zaug, 1986, The Tetrahymena ribozyme acts like an RNA restriction endonuclease, Nature 324(6096):429-33.
Zhang, 1993, Substrate specificity of the protein tyrosine phosphatases, PNAS 90: 4446-4450.
Zhang, 1999, A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays, Journal of Biomolecular Screening, 4(2): 67-73.
Zhao, 1998, Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat Biotechnol 16 (3):258-61.
Zhao, 2002, Control and Applications of Immiscible Liquids in Microchannels, J. Am. Chem. Soc, vol. 124:5284-5285.
Zheng, 2003, Screening of Protein Crystallization Conditions on a Microfluidic Chip Using Nanoliter-Size Droplets, J Am Chem Soc 125(37):11170-11171.
Zheng, 2004, A Droplet-Based, Composite PDMS/Glass Capillary Microfluidic System for Evaluating Protein Crystallization Conditions by Microbatch and Vapor-Diffusion Methods with On-Chip X-Ray Diffraction, Angew. Chem.,116:1-4.
Zheng, 2004, Formation of Droplets of Alternating Composition in Microfluidic Channels and Applications to Indexing of Concentrations in Droplet-Based /Assays, Anal. Chem.,76: 4977-4982.
Zheng, 2005, A Microiuidic Approach for Screening Submicroliter Volumes against Multiple Reagents by Using Performed Arrays of Nanoliter Plugs in a Three-Phase Liquid/Liquid/Gas Flow, Angew. Chem. Int. Ed., 44(17): 2520-2523.
Zhong, 2011, Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR, Lab on a Chip 11 (13):2167-2174.
Zimmermann, 1974, Dielectric Breakdown of Cell Membranes, Biophys J 14(11):881-889.
Zimmermann, 1992, Microscale Production of Hybridomas by Hypo-Osmolar Electrofusion, Hum. Antibod. Hybridomas, 3(1): 14-18.
Zimmermann, Bernhard G., et al., Digital PCR: a powerful new tool for noninvasive prenatal diagnosis?, Prenat Diagn 28, 1087-1093 (2008).
Zubay, 1973, In vitro synthesis of protein in microbial systems, Annu Rev Genet, 7: 267-87.
Zubay, 1980, The isolation and properties of CAP, the catabolite gene activator, Methods Enzymol, 65: 856-77.
Zuckermann, 1987, Efficient Methods for Attachment of Thiol-Specific Probes to the 3-end of Synthetic Oligodeoxyribonucleotides, Nucleic Acids Res. 15:5305-5321.

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